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CTNNBL1 associates with the NLS region of CDC5L. A , immunoprecipitation of endogenous CDC5L from 293T cells (using increasing volumes in microliters of anti-CDC5L antibody) brings down endogenous CTNNBL1 and Prp19 ( left panels ); immunoprecipitation of FLAG-tagged CTNNBL1 from 293T[FLAG-CTNNBL1] transfectants (+) but not control 293T cells (−) brings down endogenous CDC5L and Prp19 ( right panels ). IP , samples analyzed following immunoprecipitation by Western blotting ( W ) with anti-CTNNBL1, anti-CDC5L, anti-Prp19 or anti-FLAG antibodies as indicated. Lys , an aliquot (7%) of the total cell lysate probed as a control. B , the 4×NLS domain of CDC5L is required for interaction with CTNNBL1. Lysates of 293T cells that had been transfected with HA-tagged deletion mutants of CDC5L together with (+) or without (−) FLAG-tagged CTNNBL1 were subjected to immunoprecipitation with anti-FLAG-agarose followed by Western blotting with anti-HA antibodies. The upper panels show the Western blots of the immunoprecipitated material ( IP ); the lower pairs of panels show the blots of the whole cell lysates ( Lys ). The migration positions of prestained molecular weight marker proteins are indicated. C , purification of the endogenous CTNNBL1 from 293T cell extracts was accomplished by fishing with recombinant GST-NLS fusion proteins. Computationally predicted NLS sequences from CDC5L were expressed as GST fusion proteins in E. coli and immobilized on <t>glutathione-Sepharose.</t> After incubation with 293T cell lysates, total Sepharose-bound proteins were subjected to SDS-PAGE and stained with Ponceau S prior to Western blotting ( W ) with anti-CTNNBL1 antiserum. D , nuclear targeting activity of predicted CDC5L NLS sequences was tested using confocal microscopy to analyze the intracellular localization of NLS-β-galactosidase-GFP fusion proteins transfected into 293T cells. Nuclei were counterstained with DAPI. The percentage of fluorescent cells exhibiting clear nuclear fluorescence is indicated. 100 cells transfected with each construct were counted. The mean ± S.D. were calculated by analyzing at least 30 cells transduced with each construct from multiple independently acquired fields. E , direct interaction between GST-NLS fusion proteins and His-CTNNBL1 was assessed by incubating GST-NLS fusion proteins that had been bound to glutathione-Sepharose with purified His-CTNNBL1. Total Sepharose-bound proteins were stained with Ponceau S following SDS-PAGE prior to Western blotting ( W ) with anti-CTNNBL1 mAb. The purity of the recombinant His-tagged CTNNBL1 used for the binding assays is illustrated.
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Article Title:

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.208769

CTNNBL1 associates with the NLS region of CDC5L. A , immunoprecipitation of endogenous CDC5L from 293T cells (using increasing volumes in microliters of anti-CDC5L antibody) brings down endogenous CTNNBL1 and Prp19 ( left panels ); immunoprecipitation of FLAG-tagged CTNNBL1 from 293T[FLAG-CTNNBL1] transfectants (+) but not control 293T cells (−) brings down endogenous CDC5L and Prp19 ( right panels ). IP , samples analyzed following immunoprecipitation by Western blotting ( W ) with anti-CTNNBL1, anti-CDC5L, anti-Prp19 or anti-FLAG antibodies as indicated. Lys , an aliquot (7%) of the total cell lysate probed as a control. B , the 4×NLS domain of CDC5L is required for interaction with CTNNBL1. Lysates of 293T cells that had been transfected with HA-tagged deletion mutants of CDC5L together with (+) or without (−) FLAG-tagged CTNNBL1 were subjected to immunoprecipitation with anti-FLAG-agarose followed by Western blotting with anti-HA antibodies. The upper panels show the Western blots of the immunoprecipitated material ( IP ); the lower pairs of panels show the blots of the whole cell lysates ( Lys ). The migration positions of prestained molecular weight marker proteins are indicated. C , purification of the endogenous CTNNBL1 from 293T cell extracts was accomplished by fishing with recombinant GST-NLS fusion proteins. Computationally predicted NLS sequences from CDC5L were expressed as GST fusion proteins in E. coli and immobilized on glutathione-Sepharose. After incubation with 293T cell lysates, total Sepharose-bound proteins were subjected to SDS-PAGE and stained with Ponceau S prior to Western blotting ( W ) with anti-CTNNBL1 antiserum. D , nuclear targeting activity of predicted CDC5L NLS sequences was tested using confocal microscopy to analyze the intracellular localization of NLS-β-galactosidase-GFP fusion proteins transfected into 293T cells. Nuclei were counterstained with DAPI. The percentage of fluorescent cells exhibiting clear nuclear fluorescence is indicated. 100 cells transfected with each construct were counted. The mean ± S.D. were calculated by analyzing at least 30 cells transduced with each construct from multiple independently acquired fields. E , direct interaction between GST-NLS fusion proteins and His-CTNNBL1 was assessed by incubating GST-NLS fusion proteins that had been bound to glutathione-Sepharose with purified His-CTNNBL1. Total Sepharose-bound proteins were stained with Ponceau S following SDS-PAGE prior to Western blotting ( W ) with anti-CTNNBL1 mAb. The purity of the recombinant His-tagged CTNNBL1 used for the binding assays is illustrated.
Figure Legend Snippet: CTNNBL1 associates with the NLS region of CDC5L. A , immunoprecipitation of endogenous CDC5L from 293T cells (using increasing volumes in microliters of anti-CDC5L antibody) brings down endogenous CTNNBL1 and Prp19 ( left panels ); immunoprecipitation of FLAG-tagged CTNNBL1 from 293T[FLAG-CTNNBL1] transfectants (+) but not control 293T cells (−) brings down endogenous CDC5L and Prp19 ( right panels ). IP , samples analyzed following immunoprecipitation by Western blotting ( W ) with anti-CTNNBL1, anti-CDC5L, anti-Prp19 or anti-FLAG antibodies as indicated. Lys , an aliquot (7%) of the total cell lysate probed as a control. B , the 4×NLS domain of CDC5L is required for interaction with CTNNBL1. Lysates of 293T cells that had been transfected with HA-tagged deletion mutants of CDC5L together with (+) or without (−) FLAG-tagged CTNNBL1 were subjected to immunoprecipitation with anti-FLAG-agarose followed by Western blotting with anti-HA antibodies. The upper panels show the Western blots of the immunoprecipitated material ( IP ); the lower pairs of panels show the blots of the whole cell lysates ( Lys ). The migration positions of prestained molecular weight marker proteins are indicated. C , purification of the endogenous CTNNBL1 from 293T cell extracts was accomplished by fishing with recombinant GST-NLS fusion proteins. Computationally predicted NLS sequences from CDC5L were expressed as GST fusion proteins in E. coli and immobilized on glutathione-Sepharose. After incubation with 293T cell lysates, total Sepharose-bound proteins were subjected to SDS-PAGE and stained with Ponceau S prior to Western blotting ( W ) with anti-CTNNBL1 antiserum. D , nuclear targeting activity of predicted CDC5L NLS sequences was tested using confocal microscopy to analyze the intracellular localization of NLS-β-galactosidase-GFP fusion proteins transfected into 293T cells. Nuclei were counterstained with DAPI. The percentage of fluorescent cells exhibiting clear nuclear fluorescence is indicated. 100 cells transfected with each construct were counted. The mean ± S.D. were calculated by analyzing at least 30 cells transduced with each construct from multiple independently acquired fields. E , direct interaction between GST-NLS fusion proteins and His-CTNNBL1 was assessed by incubating GST-NLS fusion proteins that had been bound to glutathione-Sepharose with purified His-CTNNBL1. Total Sepharose-bound proteins were stained with Ponceau S following SDS-PAGE prior to Western blotting ( W ) with anti-CTNNBL1 mAb. The purity of the recombinant His-tagged CTNNBL1 used for the binding assays is illustrated.

Techniques Used: Immunoprecipitation, Western Blot, Transfection, Migration, Molecular Weight, Marker, Purification, Recombinant, Incubation, SDS Page, Staining, Activity Assay, Confocal Microscopy, Fluorescence, Construct, Transduction, Binding Assay

Bipartite NLS in the CTNNBL1 N-terminal region binds karyopherin α. A , direct interaction between purified karyopherin α1/2 and GST fusion proteins that contained either the indicated segments of the CTNNBL1 N-terminal region or the SV40 or nucleoplasmin ( Nplmn ) NLSs assessed by incubating the GST-NLS fusion proteins that had been bound to glutathione-Sepharose with purified His-tagged karyopherin. The karyopherin α and GST fusion proteins bound to the Sepharose were visualized with Coomassie following SDS-PAGE. B , mutational analysis of the interaction between karyopherin α1/2 and a GST fusion containing CTNNBL1 residues 13–33, with mutations at Arg 13 , Lys 16 , or Lys 17 as indicated. C , residues 1–32 of CTNNBL1 confer nuclear localization on a CTNNBL1-β-galactosidase-GFP chimera. Nuclei were counterstained with DAPI and cell membranes with wheat germ agglutinin ( WGA )-Alexa Fluor 568. D , sequence of the N-terminal region of CTNNBL1 with the bipartite NLS underlined .
Figure Legend Snippet: Bipartite NLS in the CTNNBL1 N-terminal region binds karyopherin α. A , direct interaction between purified karyopherin α1/2 and GST fusion proteins that contained either the indicated segments of the CTNNBL1 N-terminal region or the SV40 or nucleoplasmin ( Nplmn ) NLSs assessed by incubating the GST-NLS fusion proteins that had been bound to glutathione-Sepharose with purified His-tagged karyopherin. The karyopherin α and GST fusion proteins bound to the Sepharose were visualized with Coomassie following SDS-PAGE. B , mutational analysis of the interaction between karyopherin α1/2 and a GST fusion containing CTNNBL1 residues 13–33, with mutations at Arg 13 , Lys 16 , or Lys 17 as indicated. C , residues 1–32 of CTNNBL1 confer nuclear localization on a CTNNBL1-β-galactosidase-GFP chimera. Nuclei were counterstained with DAPI and cell membranes with wheat germ agglutinin ( WGA )-Alexa Fluor 568. D , sequence of the N-terminal region of CTNNBL1 with the bipartite NLS underlined .

Techniques Used: Purification, SDS Page, Whole Genome Amplification, Sequencing

AID nuclear import mutants show diminished CTNNBL1 binding. A , subcellular distribution of chimeric AID[F193A]-β-galactosidase-GFP fusion proteins. AID[F193A]-β-galactosidase-GFP proteins with the indicated point mutations were expressed in 293T cells and visualized by confocal microscopy. Nuclei were counterstained with DAPI. The F193A mutation in the reporter protein interferes with the AID nuclear export sequence ( 28 , 29 ), allowing import to be monitored. The percentage of fluorescent cells exhibiting clear nuclear fluorescence is indicated. 30 cells transfected with each construct were counted. The mean ± S.D. calculated from three independent experiments are indicated. B , diminished interaction of AID nuclear import mutants with CTNNBL1. GST-tagged AID and point mutants thereof expressed in E. coli and bound onto glutathione-Sepharose were incubated with extracts of 293T cells that had been transfected with FLAG-CTNNBL1. Following SDS-PAGE, the abundance of GST-AID proteins was demonstrated by staining with Coomassie; bound FLAG-CTNNBL1 was detected by Western blotting ( W ) with anti-FLAG antibody. C , R24W and V18S,R19V mutations in AID affecting binding to both CTNNBL1 ( top panels ) and karyopherin α1 ( lower panels ). Binding of GST-AID mutants to FLAG-CTNNBL1 and HA-tagged karyopherin α1 was assessed as in B , except using ant-HA antibody to detect bound HA-karyopherin α1.
Figure Legend Snippet: AID nuclear import mutants show diminished CTNNBL1 binding. A , subcellular distribution of chimeric AID[F193A]-β-galactosidase-GFP fusion proteins. AID[F193A]-β-galactosidase-GFP proteins with the indicated point mutations were expressed in 293T cells and visualized by confocal microscopy. Nuclei were counterstained with DAPI. The F193A mutation in the reporter protein interferes with the AID nuclear export sequence ( 28 , 29 ), allowing import to be monitored. The percentage of fluorescent cells exhibiting clear nuclear fluorescence is indicated. 30 cells transfected with each construct were counted. The mean ± S.D. calculated from three independent experiments are indicated. B , diminished interaction of AID nuclear import mutants with CTNNBL1. GST-tagged AID and point mutants thereof expressed in E. coli and bound onto glutathione-Sepharose were incubated with extracts of 293T cells that had been transfected with FLAG-CTNNBL1. Following SDS-PAGE, the abundance of GST-AID proteins was demonstrated by staining with Coomassie; bound FLAG-CTNNBL1 was detected by Western blotting ( W ) with anti-FLAG antibody. C , R24W and V18S,R19V mutations in AID affecting binding to both CTNNBL1 ( top panels ) and karyopherin α1 ( lower panels ). Binding of GST-AID mutants to FLAG-CTNNBL1 and HA-tagged karyopherin α1 was assessed as in B , except using ant-HA antibody to detect bound HA-karyopherin α1.

Techniques Used: Binding Assay, Confocal Microscopy, Mutagenesis, Sequencing, Fluorescence, Transfection, Construct, Incubation, SDS Page, Staining, Western Blot

Proteins associated with immunopurified FLAG-CTNNBL1. A , proteins bound to FLAG-CTNNBL1 or FLAG-APOBEC2 control were immunopurified using anti-FLAG M2-agarose, eluted with 3×FLAG peptide, separated by SDS-PAGE, and stained with Coomassie. Prestained molecular weight markers were run alongside the samples. B , proteins co-immunopurifying with FLAG-CTNNBL1 were subjected to tryptic digestion and mass spectrometric identification. All proteins identified with a Mowse score > 65 and which were not also identified in the FLAG-APOBEC2 control sample ( e.g. keratin; immunoglobulin fragments) are shown.
Figure Legend Snippet: Proteins associated with immunopurified FLAG-CTNNBL1. A , proteins bound to FLAG-CTNNBL1 or FLAG-APOBEC2 control were immunopurified using anti-FLAG M2-agarose, eluted with 3×FLAG peptide, separated by SDS-PAGE, and stained with Coomassie. Prestained molecular weight markers were run alongside the samples. B , proteins co-immunopurifying with FLAG-CTNNBL1 were subjected to tryptic digestion and mass spectrometric identification. All proteins identified with a Mowse score > 65 and which were not also identified in the FLAG-APOBEC2 control sample ( e.g. keratin; immunoglobulin fragments) are shown.

Techniques Used: SDS Page, Staining, Molecular Weight

CTNNBL1 binds karyopherin α (but not β1) through its N-terminal region. A , immunoprecipitation of HA-karyopherin α1 (but not HA-tagged karyopherin β1, Crm1, or Ran) bringing down FLAG-CTNNBL1 from co-transfected 293T cell lysates. IP , samples subjected to immunoprecipitation on anti-HA-agarose were analyzed by Western blotting ( W ) with anti-FLAG antibody. Lys , Western blots of samples of total cell lysates were probed with anti-HA or anti-FLAG antibodies as a control. B , in vitro interaction between purified His-CTNNBL1 and purified GST-karyopherin αs. GST-tagged nuclear transport factors expressed in E. coli and bound onto glutathione-Sepharose were incubated with recombinant His-CTNNBL1. Following SDS-PAGE, bound CTNNBL1 was detected by Western blotting with anti-CTNNBL1 mAb ( top panel ) whereas total Sepharose-bound proteins were identified by Coomassie staining ( middle panel ). SDS-PAGE analysis of total input GST-fusion protein is shown in the bottom panel . The right lane in the top two panels shows the migration of purified His-CTNNBL1. Snptn , snurportin. C , the CTNNBL1 N-terminal region is required for interaction with karyopherin αs. GST-karyopherin α1/α2 bound to glutathione-Sepharose was incubated with purified His-CTNNBL1 or His-ΔCTNNBL1 (lacking the N-terminal 76 amino acids). Sepharose-bound proteins ( top panel ) as well as samples of the supernatants from the incubation (lower panel) were separated on SDS-PAGE and stained with Coomassie; aliquots of the input His-CTNNBL1 and His-ΔCTNNBL1 are shown in the two right lanes .
Figure Legend Snippet: CTNNBL1 binds karyopherin α (but not β1) through its N-terminal region. A , immunoprecipitation of HA-karyopherin α1 (but not HA-tagged karyopherin β1, Crm1, or Ran) bringing down FLAG-CTNNBL1 from co-transfected 293T cell lysates. IP , samples subjected to immunoprecipitation on anti-HA-agarose were analyzed by Western blotting ( W ) with anti-FLAG antibody. Lys , Western blots of samples of total cell lysates were probed with anti-HA or anti-FLAG antibodies as a control. B , in vitro interaction between purified His-CTNNBL1 and purified GST-karyopherin αs. GST-tagged nuclear transport factors expressed in E. coli and bound onto glutathione-Sepharose were incubated with recombinant His-CTNNBL1. Following SDS-PAGE, bound CTNNBL1 was detected by Western blotting with anti-CTNNBL1 mAb ( top panel ) whereas total Sepharose-bound proteins were identified by Coomassie staining ( middle panel ). SDS-PAGE analysis of total input GST-fusion protein is shown in the bottom panel . The right lane in the top two panels shows the migration of purified His-CTNNBL1. Snptn , snurportin. C , the CTNNBL1 N-terminal region is required for interaction with karyopherin αs. GST-karyopherin α1/α2 bound to glutathione-Sepharose was incubated with purified His-CTNNBL1 or His-ΔCTNNBL1 (lacking the N-terminal 76 amino acids). Sepharose-bound proteins ( top panel ) as well as samples of the supernatants from the incubation (lower panel) were separated on SDS-PAGE and stained with Coomassie; aliquots of the input His-CTNNBL1 and His-ΔCTNNBL1 are shown in the two right lanes .

Techniques Used: Immunoprecipitation, Transfection, Western Blot, In Vitro, Purification, Incubation, Recombinant, SDS Page, Staining, Migration

CTNNBL1 binds Prp31 through its NLS. A , selective nuclear accumulation of Prp31-GFP but not Prp31[ΔNLS]-GFP in transfected 293T cells was assessed by confocal microscopy. Nuclei were counterstained with DAPI. B , Prp31-GFP ( WT ) but not Prp31[ΔNLS]-GFP (ΔNLS) associates with FLAG-tagged CTNNBL1 in co-transfected 293T cells. IP , GFP-tagged Prp31 or Prp31[ΔNLS] was purified from the lysates using anti-GFP antibody and protein A-Sepharose, subjected to SDS-PAGE and Western blotted ( W ) with anti-FLAG antibody. Lys , samples of total cell lysates were Western blotted with ant-GFP or anti-FLAG antibodies as expression controls. C , GST-[Prp31NLS] fusion protein pulls down endogenous CTNNBL1 from a 293T whole cell lysate. The indicated GST-NLS chimeric proteins (see Fig. 4 C for NLS sequences) that had been purified from E. coli and bound to glutathione-Sepharose were incubated with 293T cell lysates. Total Sepharose-bound proteins were stained with Ponceau S following SDS-PAGE prior to Western blotting ( W ) with anti-CTNNBL1 antiserum. D , direct interaction between GST-[Prp31NLS] (as well as other GST-NLS) fusion proteins and His-CTNNBL1 was assessed by incubating GST-NLS fusion proteins that had been bound to glutathione-Sepharose with purified His-CTNNBL1. Bound proteins were stained with Ponceau S prior to probing with anti-CTNNBL1 mAb. An aliquot of the input CTNNBL1 is shown in the right lane .
Figure Legend Snippet: CTNNBL1 binds Prp31 through its NLS. A , selective nuclear accumulation of Prp31-GFP but not Prp31[ΔNLS]-GFP in transfected 293T cells was assessed by confocal microscopy. Nuclei were counterstained with DAPI. B , Prp31-GFP ( WT ) but not Prp31[ΔNLS]-GFP (ΔNLS) associates with FLAG-tagged CTNNBL1 in co-transfected 293T cells. IP , GFP-tagged Prp31 or Prp31[ΔNLS] was purified from the lysates using anti-GFP antibody and protein A-Sepharose, subjected to SDS-PAGE and Western blotted ( W ) with anti-FLAG antibody. Lys , samples of total cell lysates were Western blotted with ant-GFP or anti-FLAG antibodies as expression controls. C , GST-[Prp31NLS] fusion protein pulls down endogenous CTNNBL1 from a 293T whole cell lysate. The indicated GST-NLS chimeric proteins (see Fig. 4 C for NLS sequences) that had been purified from E. coli and bound to glutathione-Sepharose were incubated with 293T cell lysates. Total Sepharose-bound proteins were stained with Ponceau S following SDS-PAGE prior to Western blotting ( W ) with anti-CTNNBL1 antiserum. D , direct interaction between GST-[Prp31NLS] (as well as other GST-NLS) fusion proteins and His-CTNNBL1 was assessed by incubating GST-NLS fusion proteins that had been bound to glutathione-Sepharose with purified His-CTNNBL1. Bound proteins were stained with Ponceau S prior to probing with anti-CTNNBL1 mAb. An aliquot of the input CTNNBL1 is shown in the right lane .

Techniques Used: Transfection, Confocal Microscopy, Purification, SDS Page, Western Blot, Expressing, Incubation, Staining

2) Product Images from "Identification of Yin-Yang Regulators and a Phosphorylation Consensus for Male Germ Cell-Associated Kinase (MAK)-Related Kinase ▿"

Article Title: Identification of Yin-Yang Regulators and a Phosphorylation Consensus for Male Germ Cell-Associated Kinase (MAK)-Related Kinase ▿

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00816-06

PP5 dephosphorylates MRK in the TDY motif in vitro and in vivo. (A) GST-MRK purified on glutathione-Sepharose beads from HEK293T cells was incubated with purified Flag-PP5 protein in the absence (−) or presence (+) of 50 μM arachidonic acid (AA) in vitro. For a control, 1 μM okadaic acid (OK) was added to inhibit PP5 phosphatase activity. After the reaction, the GST-MRK bead samples were analyzed for MRK phosphorylation in the TDY motif by Western blotting (immunoblotting [IB]) against the anti-phospho-ERK antibody (anti-pERK). The amount of GST-MRK on beads was indicated by Western blotting against the anti-MRK antibody. (B) GST-MRK was coexpressed with either the full-length Flag-PP5 or the catalytic inactive form Flag-PP5(1-159) in HEK293T cells. GST fusion proteins were purified on glutathione-Sepharose beads and analyzed for MRK phosphorylation in the TDY motif as described above for panel A. The amounts of GST-MRK, Flag-PP5, and Flag-PP5(1-159) in cell lysate were indicated by Western blotting against the anti-MRK and anti-Flag antibodies. KD GST-MRK was used as a negative control for TDY motif phosphorylation. (C) GST-MRK was coexpressed with either the full-length Flag-PP5 or the catalytic inactive form Flag-PP5(1-159) in HEK293T cells. GST fusion proteins were analyzed for MRK phosphorylation in the TDY motif as described above for panel B and for MRK kinase activity in vitro using MBP as the substrate.
Figure Legend Snippet: PP5 dephosphorylates MRK in the TDY motif in vitro and in vivo. (A) GST-MRK purified on glutathione-Sepharose beads from HEK293T cells was incubated with purified Flag-PP5 protein in the absence (−) or presence (+) of 50 μM arachidonic acid (AA) in vitro. For a control, 1 μM okadaic acid (OK) was added to inhibit PP5 phosphatase activity. After the reaction, the GST-MRK bead samples were analyzed for MRK phosphorylation in the TDY motif by Western blotting (immunoblotting [IB]) against the anti-phospho-ERK antibody (anti-pERK). The amount of GST-MRK on beads was indicated by Western blotting against the anti-MRK antibody. (B) GST-MRK was coexpressed with either the full-length Flag-PP5 or the catalytic inactive form Flag-PP5(1-159) in HEK293T cells. GST fusion proteins were purified on glutathione-Sepharose beads and analyzed for MRK phosphorylation in the TDY motif as described above for panel A. The amounts of GST-MRK, Flag-PP5, and Flag-PP5(1-159) in cell lysate were indicated by Western blotting against the anti-MRK and anti-Flag antibodies. KD GST-MRK was used as a negative control for TDY motif phosphorylation. (C) GST-MRK was coexpressed with either the full-length Flag-PP5 or the catalytic inactive form Flag-PP5(1-159) in HEK293T cells. GST fusion proteins were analyzed for MRK phosphorylation in the TDY motif as described above for panel B and for MRK kinase activity in vitro using MBP as the substrate.

Techniques Used: In Vitro, In Vivo, Purification, Incubation, Activity Assay, Western Blot, Negative Control

Recombinant MRK interacts with recombinant Scythe in cells and phosphorylates recombinant Scythe in vitro. (A) GST-MRK or GST was coexpressed with Flag-Scythe in HEK293T cells. GST fusion proteins were pulled down by glutathione-Sepharose beads and analyzed for association with Flag-Scythe by Western blotting (immunoblotting [IB]) against the anti-Flag antibody. The amounts of GST, GST-MRK and Flag-Scythe in cell lysate were indicated by Western blotting. The positions of molecular mass markers (in kilodaltons) are indicated to the left of the blot. (B) Flag-Scythe immunoprecipitated from HEK293T cells was assayed for phosphorylation by MRK in vitro using purified His-MRK(1-296) (top panel). MBP served as a positive-control substrate for MRK kinase activity (bottom panel).
Figure Legend Snippet: Recombinant MRK interacts with recombinant Scythe in cells and phosphorylates recombinant Scythe in vitro. (A) GST-MRK or GST was coexpressed with Flag-Scythe in HEK293T cells. GST fusion proteins were pulled down by glutathione-Sepharose beads and analyzed for association with Flag-Scythe by Western blotting (immunoblotting [IB]) against the anti-Flag antibody. The amounts of GST, GST-MRK and Flag-Scythe in cell lysate were indicated by Western blotting. The positions of molecular mass markers (in kilodaltons) are indicated to the left of the blot. (B) Flag-Scythe immunoprecipitated from HEK293T cells was assayed for phosphorylation by MRK in vitro using purified His-MRK(1-296) (top panel). MBP served as a positive-control substrate for MRK kinase activity (bottom panel).

Techniques Used: Recombinant, In Vitro, Western Blot, Immunoprecipitation, Purification, Positive Control, Activity Assay

CCRK, but not the Cdk7 complex, increases MRK phosphorylation in the TDY motif in cells. (A) Alignment of the T-loops of MRK, MAK, and their putative homologs in yeasts, Ime2p, Pit1, and Mde3. Identical amino acids are shown in bold type, and the TDY motif is underlined. (B and C) GST-MRK was coexpressed with either WT and KD Flag-CCRK (B) or recombinant Cdk7/cyclin H/MAT1 complex (C) in HEK293T cells. GST fusion proteins were pulled down by glutathione-Sepharose beads and analyzed for MRK phosphorylation in the TDY motif by Western blotting (immunoblotting [IB]) against the anti-phospho-ERK antibody (anti-pERK). The amounts of recombinant proteins on beads and in cell lysate were indicated by Western blotting. After coexpression with either WT or KD Flag-CCRK, GST-MRK bead samples were assayed for kinase activity in vitro using myelin basic protein as the substrate (B). Endo+Exo, endogenous and exogenous.
Figure Legend Snippet: CCRK, but not the Cdk7 complex, increases MRK phosphorylation in the TDY motif in cells. (A) Alignment of the T-loops of MRK, MAK, and their putative homologs in yeasts, Ime2p, Pit1, and Mde3. Identical amino acids are shown in bold type, and the TDY motif is underlined. (B and C) GST-MRK was coexpressed with either WT and KD Flag-CCRK (B) or recombinant Cdk7/cyclin H/MAT1 complex (C) in HEK293T cells. GST fusion proteins were pulled down by glutathione-Sepharose beads and analyzed for MRK phosphorylation in the TDY motif by Western blotting (immunoblotting [IB]) against the anti-phospho-ERK antibody (anti-pERK). The amounts of recombinant proteins on beads and in cell lysate were indicated by Western blotting. After coexpression with either WT or KD Flag-CCRK, GST-MRK bead samples were assayed for kinase activity in vitro using myelin basic protein as the substrate (B). Endo+Exo, endogenous and exogenous.

Techniques Used: Recombinant, Western Blot, Activity Assay, In Vitro

Recombinant CCRK interacts with recombinant MRK in cells and phosphorylates recombinant MRK in vitro. (A) GST-MRK or GST was coexpressed with Flag-CCRK in HEK293T cells. GST fusion proteins were pulled down by glutathione-Sepharose beads and analyzed for association with Flag-CCRK by Western blotting (immunoblotting [IB]) against the anti-Flag antibody. The amounts of GST, GST-MRK, and Flag-CCRK in cell lysate were indicated by Western blotting against the anti-GST and anti-Flag antibodies. The positions of molecular mass markers (in kilodaltons) are shown to the left of the blot. (B) GST-MRK(1-300) purified from E. coli was assayed for in vitro phosphorylation by WT or KD Flag-CCRK purified from HEK293T cells. MRK phosphorylation (autoradiograph) and the amounts of substrates and kinases (Coomassie blue-stained gels) are shown.
Figure Legend Snippet: Recombinant CCRK interacts with recombinant MRK in cells and phosphorylates recombinant MRK in vitro. (A) GST-MRK or GST was coexpressed with Flag-CCRK in HEK293T cells. GST fusion proteins were pulled down by glutathione-Sepharose beads and analyzed for association with Flag-CCRK by Western blotting (immunoblotting [IB]) against the anti-Flag antibody. The amounts of GST, GST-MRK, and Flag-CCRK in cell lysate were indicated by Western blotting against the anti-GST and anti-Flag antibodies. The positions of molecular mass markers (in kilodaltons) are shown to the left of the blot. (B) GST-MRK(1-300) purified from E. coli was assayed for in vitro phosphorylation by WT or KD Flag-CCRK purified from HEK293T cells. MRK phosphorylation (autoradiograph) and the amounts of substrates and kinases (Coomassie blue-stained gels) are shown.

Techniques Used: Recombinant, In Vitro, Western Blot, Purification, Autoradiography, Staining

Recombinant MRK interacts with both recombinant and endogenous PP5 in cells. GST-MRK or GST was expressed alone or coexpressed with Flag-PP5 in HEK293T cells. GST fusion proteins were purified on glutathione-Sepharose beads and analyzed for association with recombinant and/or endogenous PP5 by Western blotting (immunoblotting [IB]) against the anti-Flag and anti-PP5 antibodies, respectively. The amounts of GST fusion proteins on beads and the amounts of Flag-PP5 and endogenous PP5 in cell lysate were indicated by Western blotting.
Figure Legend Snippet: Recombinant MRK interacts with both recombinant and endogenous PP5 in cells. GST-MRK or GST was expressed alone or coexpressed with Flag-PP5 in HEK293T cells. GST fusion proteins were purified on glutathione-Sepharose beads and analyzed for association with recombinant and/or endogenous PP5 by Western blotting (immunoblotting [IB]) against the anti-Flag and anti-PP5 antibodies, respectively. The amounts of GST fusion proteins on beads and the amounts of Flag-PP5 and endogenous PP5 in cell lysate were indicated by Western blotting.

Techniques Used: Recombinant, Purification, Western Blot

Activation of endogenous PP5 by H 2 O 2 -induced oxidative stress causes dephosphorylation in the TDY motif of MRK. HEK293T cells transfected with GST-MRK were treated either with an increasing concentration of H 2 O 2 (0 to 5 mM) for 45 min (A) or with 5 mM H 2 O 2 for an increasing length of time (0 to 45 min) (B) before harvesting. GST fusion proteins were pulled down by glutathione-Sepharose beads and analyzed for MRK phosphorylation in the TDY motif by Western blotting (immunoblotting [IB]) against the anti-phospho-ERK antibody (pERK). The amounts of GST-MRK on beads were indicated by Western blotting against the anti-GST antibody. The amounts of endogenous full-length PP5 (55 kDa) and the C-terminally truncated form of PP5 (50 kDa) in cell lysate and on beads were indicated by the anti-PP5 antibody. For comparison, the TEY motif phosphorylation of ERK1 and ERK2 under oxidative stress was also analyzed by the anti-phospho-ERK antibody (B).
Figure Legend Snippet: Activation of endogenous PP5 by H 2 O 2 -induced oxidative stress causes dephosphorylation in the TDY motif of MRK. HEK293T cells transfected with GST-MRK were treated either with an increasing concentration of H 2 O 2 (0 to 5 mM) for 45 min (A) or with 5 mM H 2 O 2 for an increasing length of time (0 to 45 min) (B) before harvesting. GST fusion proteins were pulled down by glutathione-Sepharose beads and analyzed for MRK phosphorylation in the TDY motif by Western blotting (immunoblotting [IB]) against the anti-phospho-ERK antibody (pERK). The amounts of GST-MRK on beads were indicated by Western blotting against the anti-GST antibody. The amounts of endogenous full-length PP5 (55 kDa) and the C-terminally truncated form of PP5 (50 kDa) in cell lysate and on beads were indicated by the anti-PP5 antibody. For comparison, the TEY motif phosphorylation of ERK1 and ERK2 under oxidative stress was also analyzed by the anti-phospho-ERK antibody (B).

Techniques Used: Activation Assay, De-Phosphorylation Assay, Transfection, Concentration Assay, Western Blot

3) Product Images from "E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue"

Article Title: E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue

Journal: PLoS ONE

doi: 10.1371/journal.pone.0163710

The Residue Cys118 Plays a Key Role in the Autoubiquitination of UCP. (A) An autoubiquitination assay was performed using His-UCP WT and His-UCP C118A (0.2 μg each) along with wild-type ubiquitin (Ub WT ) at various time points. Ubiquitinated forms were detected by immunoblotting using anti-ubiquitin antibody. (B) An autoubiquitination assay was performed using His-UCP WT and His-UCP C118A (0.2 μg each) along with lysine-null ubiquitin (Ub ∆K ) at various time points. Ubiquitinated forms were detected by immunoblotting using anti-ubiquitin antibody. (C) In vitro ubiquitination assay was performed using His-UCP WT (0.2 μg) and GST-UCP C95A (2 μg) at various time points. GST-UCP C95A was pulled down using GST agarose and then polyubiquitination was separated by SDS-PAGE under denaturing or non-denaturing (without β-mercaptoethanol) condition. The polyubiquitin chains were detected by anti-Flag antibody. (D) Illustration of the expected reaction steps for polyubiquitination by UCP in the trans manner and the status of polyubiquitin chains on the substrate under different conditions. (E) An in vitro ubiquitination assay was performed using wild-type UCP (His-UCP WT ), double-mutant UCP with K78R and K100R (His-UCP K76R/K100R ), UCP-C118A mutant (His-UCP C118A ) (0.2 μg each) and GST-UCP C95A (2 μg) at 37°C for 1 h in reaction buffer. After the reaction, GST-UCP C95A was pulled down with GST agarose, and polyubiquitination was analyzed by immunoblotting under denaturing (+β-mercaptoethanol) or non-denaturing (-β-mercaptoethanol) conditions using anti-Flag antibody.
Figure Legend Snippet: The Residue Cys118 Plays a Key Role in the Autoubiquitination of UCP. (A) An autoubiquitination assay was performed using His-UCP WT and His-UCP C118A (0.2 μg each) along with wild-type ubiquitin (Ub WT ) at various time points. Ubiquitinated forms were detected by immunoblotting using anti-ubiquitin antibody. (B) An autoubiquitination assay was performed using His-UCP WT and His-UCP C118A (0.2 μg each) along with lysine-null ubiquitin (Ub ∆K ) at various time points. Ubiquitinated forms were detected by immunoblotting using anti-ubiquitin antibody. (C) In vitro ubiquitination assay was performed using His-UCP WT (0.2 μg) and GST-UCP C95A (2 μg) at various time points. GST-UCP C95A was pulled down using GST agarose and then polyubiquitination was separated by SDS-PAGE under denaturing or non-denaturing (without β-mercaptoethanol) condition. The polyubiquitin chains were detected by anti-Flag antibody. (D) Illustration of the expected reaction steps for polyubiquitination by UCP in the trans manner and the status of polyubiquitin chains on the substrate under different conditions. (E) An in vitro ubiquitination assay was performed using wild-type UCP (His-UCP WT ), double-mutant UCP with K78R and K100R (His-UCP K76R/K100R ), UCP-C118A mutant (His-UCP C118A ) (0.2 μg each) and GST-UCP C95A (2 μg) at 37°C for 1 h in reaction buffer. After the reaction, GST-UCP C95A was pulled down with GST agarose, and polyubiquitination was analyzed by immunoblotting under denaturing (+β-mercaptoethanol) or non-denaturing (-β-mercaptoethanol) conditions using anti-Flag antibody.

Techniques Used: In Vitro, Ubiquitin Assay, SDS Page, Mutagenesis

UCP Forms Polyubiquitin Chains on Specific Lysine Residues in its Substrate. (A) In vitro ubiquitination assays were performed using His-UCP WT as the enzyme and inactive UCP mutants containing lysine-to-arginine mutations as the substrates (GST-UCP-C95A, GST-UCP- C95A/K76R, GST-UCP-C95A/K100R, and GST-UCP-C95A/K76R,K100R) (2 μg). After the reaction, GST-UCP was pulled down with GST agarose at 4°C for 2 h. The ubiquitinated forms were visualized with anti-Flag antibody. (B) An in vitro ubiquitination assay was performed using His-UCP WT (0.2 μg) and wild-type and/or single-lysine pVHL mutants (VHL K159 , VHL K171 , VHL K196 and VHL ∆K ) (2 μg) at 37°C for 1 h. After incubation, pVHL was pulled down with GST agarose, and ubiquitinated forms were detected by immunoblotting using anti-Flag antibody. (C) Wild-type pVHL (5 μg), single-lysine pVHL (5 μg) mutant and HA-Ubiquitin plasmids (2 μg) were co-transfected into HEK-293T cells, which were then incubated with 10 μM MG132 for 12 h. After the cells were lysed, pVHL was pulled down with GST agarose, and ubiquitinated forms were detected by immunoblotting with anti-HA antibody.
Figure Legend Snippet: UCP Forms Polyubiquitin Chains on Specific Lysine Residues in its Substrate. (A) In vitro ubiquitination assays were performed using His-UCP WT as the enzyme and inactive UCP mutants containing lysine-to-arginine mutations as the substrates (GST-UCP-C95A, GST-UCP- C95A/K76R, GST-UCP-C95A/K100R, and GST-UCP-C95A/K76R,K100R) (2 μg). After the reaction, GST-UCP was pulled down with GST agarose at 4°C for 2 h. The ubiquitinated forms were visualized with anti-Flag antibody. (B) An in vitro ubiquitination assay was performed using His-UCP WT (0.2 μg) and wild-type and/or single-lysine pVHL mutants (VHL K159 , VHL K171 , VHL K196 and VHL ∆K ) (2 μg) at 37°C for 1 h. After incubation, pVHL was pulled down with GST agarose, and ubiquitinated forms were detected by immunoblotting using anti-Flag antibody. (C) Wild-type pVHL (5 μg), single-lysine pVHL (5 μg) mutant and HA-Ubiquitin plasmids (2 μg) were co-transfected into HEK-293T cells, which were then incubated with 10 μM MG132 for 12 h. After the cells were lysed, pVHL was pulled down with GST agarose, and ubiquitinated forms were detected by immunoblotting with anti-HA antibody.

Techniques Used: In Vitro, Ubiquitin Assay, Incubation, Mutagenesis, Transfection

Autoubiquitination of UCP Occurs in an E3-independent Manner. (A) An autoubiquitination assay was performed using UCP at various time points. GST-UCP (0.2 μg) was incubated at 37°C in the presence of E1 and Flag-ubiquitin, and autoubiquitination was visualized with anti-Flag antibody. (B) The catalytic ability of UncH5c proteins was analyzed in an in vitro autoubiquitination assay. GST-UbcH5c proteins (0.2 μg) and GST-UCP (0.2 μg) were incubated at 37°C for 40 min in the presence of E1 and Flag-ubiquitin, and the ubiquitinated proteins were detected by immunoblotting using anti-Flag antibody. (C) The lysine-specific linkage of UCP was defined using lysine-to-arginine ubiquitin mutants (K6R, K11R, K48R and K63R), single-lysine ubiquitin mutants (K6, K11, K48 and K63) and a lysine-null ubiquitin mutant (K-null). Autoubiquitination assays were performed using GST-UCP (0.2 μg) and wild type ubiquitin or ubiquitin mutants at 37°C for 1 h, and ubiquitinated proteins were detected by immunoblotting using anti-ubiquitin antibody. (D) GST-UCP (0.2 μg) and His-UCP C95A (2 μg) were incubated at 37°C for 1 h in the presence of E1 and Flag-ubiquitin. His-UCP C95A was then pulled down with Ni-NTA agarose, and His-UCP C95A polyubiquitination was detected by immunoblotting using anti-Flag antibody.
Figure Legend Snippet: Autoubiquitination of UCP Occurs in an E3-independent Manner. (A) An autoubiquitination assay was performed using UCP at various time points. GST-UCP (0.2 μg) was incubated at 37°C in the presence of E1 and Flag-ubiquitin, and autoubiquitination was visualized with anti-Flag antibody. (B) The catalytic ability of UncH5c proteins was analyzed in an in vitro autoubiquitination assay. GST-UbcH5c proteins (0.2 μg) and GST-UCP (0.2 μg) were incubated at 37°C for 40 min in the presence of E1 and Flag-ubiquitin, and the ubiquitinated proteins were detected by immunoblotting using anti-Flag antibody. (C) The lysine-specific linkage of UCP was defined using lysine-to-arginine ubiquitin mutants (K6R, K11R, K48R and K63R), single-lysine ubiquitin mutants (K6, K11, K48 and K63) and a lysine-null ubiquitin mutant (K-null). Autoubiquitination assays were performed using GST-UCP (0.2 μg) and wild type ubiquitin or ubiquitin mutants at 37°C for 1 h, and ubiquitinated proteins were detected by immunoblotting using anti-ubiquitin antibody. (D) GST-UCP (0.2 μg) and His-UCP C95A (2 μg) were incubated at 37°C for 1 h in the presence of E1 and Flag-ubiquitin. His-UCP C95A was then pulled down with Ni-NTA agarose, and His-UCP C95A polyubiquitination was detected by immunoblotting using anti-Flag antibody.

Techniques Used: Incubation, In Vitro, Mutagenesis

The UBC Domain of UCP can Forms Polyubiquitin Chains on the N-terminus. (A) The catalytic activity of UCP WT (0.2 μg) and truncated UCP mutants (UCP N-term , UCP Core , UCP C-term , UCP ∆N and UCP ∆C ) (0.2 μg) was assessed using an in vitro ubiquitination assay. The proteins were incubated at 37°C for 1 h in the presence of E1 and His-ubiquitin, and the ubiquitinated proteins were detected by immunoblotting using anti-His antibody. (B) The substrate region of UCP for autoubiquitination was analyzed in stable HeLa cell lines transfected with shRNA-control or shRNA-UCP. Different stable HeLa cell lines were co-transfected with plasmids encoding each truncated UCP mutant (5 μg) and HA-Ubiquitin (2 μg), treated with 10μM MG132 for 12 h and harvested at 48 h post-transfection. The cells were then lysed, and UCP was pulled down with GST-agarose. The ubiquitinated domains were subsequently detected by immunoblotting using anti-HA antibody. (C) GST-tagged UCP WT (0.2 μg) and truncated UCP mutants (UCP C-term and UCP ∆C , 0.2 μg) were mixed with His-VHL (2 μg) and subjected to an in vitro ubiquitination assays at 37°C for 1 h. pVHL was pulled down with Ni-NTA agarose, and ubiquitinated forms were detected by immunoblotting using anti-Flag antibody. (D) HA-VHL (5 μg) and GST-wild-type UCP or truncated UCP mutant plasmids (5 μg) were co-transfected into HEK-293T cells and treated with/without 10 μM MG132 for 12 h. Changes in the pVHL expression level were then detected by immunoblotting.
Figure Legend Snippet: The UBC Domain of UCP can Forms Polyubiquitin Chains on the N-terminus. (A) The catalytic activity of UCP WT (0.2 μg) and truncated UCP mutants (UCP N-term , UCP Core , UCP C-term , UCP ∆N and UCP ∆C ) (0.2 μg) was assessed using an in vitro ubiquitination assay. The proteins were incubated at 37°C for 1 h in the presence of E1 and His-ubiquitin, and the ubiquitinated proteins were detected by immunoblotting using anti-His antibody. (B) The substrate region of UCP for autoubiquitination was analyzed in stable HeLa cell lines transfected with shRNA-control or shRNA-UCP. Different stable HeLa cell lines were co-transfected with plasmids encoding each truncated UCP mutant (5 μg) and HA-Ubiquitin (2 μg), treated with 10μM MG132 for 12 h and harvested at 48 h post-transfection. The cells were then lysed, and UCP was pulled down with GST-agarose. The ubiquitinated domains were subsequently detected by immunoblotting using anti-HA antibody. (C) GST-tagged UCP WT (0.2 μg) and truncated UCP mutants (UCP C-term and UCP ∆C , 0.2 μg) were mixed with His-VHL (2 μg) and subjected to an in vitro ubiquitination assays at 37°C for 1 h. pVHL was pulled down with Ni-NTA agarose, and ubiquitinated forms were detected by immunoblotting using anti-Flag antibody. (D) HA-VHL (5 μg) and GST-wild-type UCP or truncated UCP mutant plasmids (5 μg) were co-transfected into HEK-293T cells and treated with/without 10 μM MG132 for 12 h. Changes in the pVHL expression level were then detected by immunoblotting.

Techniques Used: Activity Assay, In Vitro, Ubiquitin Assay, Incubation, Transfection, shRNA, Mutagenesis, Expressing

Intermolecular Active Cys118 Residues are Required for Autoubiquitination of UCP. (A) Autoubiquitination was assessed using GST-UCP C95A (1 μg) and GST-UCP ∆N (0.5, 1, or 2 μg), and polyubiquitination was analyzed by immunoblotting using anti-Flag antibody. (B) An in vitro ubiquitination assay was performed using the protein pair GST-UCP ∆N /GST-UCP C95A (each 0.2 μg) and His-VHL (2 μg). His-VHL protein was pulled down with Ni-NTA agarose, and ubiquitinated forms were analyzed by immunoblotting using anti-Flag antibody. (C) HA-VHL and UCP WT or UCP mutants (UCP C95A , UCP C118A and UCP ∆N ) or various pairs of UCP mutant (UCP C95A /UCP ΔN , UCP ΔN /UCP C118A ) plasmids (total of 10 μg) were co-transfected into HEK-293T cells and treated with/without 10 μM MG132 for 12 h. At 48 h post-transfection, the cells were harvested and lysed. Changes in pVHL expression levels were detected by immunoblotting. (D) An in vitro ubiquitination assay was performed using wild-type UCP (0.2 μg) or UCP mutants (GST-UCP C118A and GST-UCP ∆N , each 0.2 μg) and His-UCP C95A (2 μg). His-UCP C95A protein was pulled down with Ni-NTA agarose, and ubiquitinated forms of His-UCP C95A were assessed by immunoblotting using anti-Flag antibody. (E) An in vitro ubiquitination assay was performed using wild-type UCP (0.2 μg), double-mutant UCP with K76R and K100R (His-UCP K76R/K100R, 0.2 μg), C118A-mutant UCP (His-UCP C118A, 0.2 μg) and GST-UCP C95A or GST-UCP CA (2 μg) at 37°C for 1 h. The GST-UCP C95A or GST-UCP CA protein was pulled down with GST agarose and analyzed by immunoblotting using anti-Flag antibody.
Figure Legend Snippet: Intermolecular Active Cys118 Residues are Required for Autoubiquitination of UCP. (A) Autoubiquitination was assessed using GST-UCP C95A (1 μg) and GST-UCP ∆N (0.5, 1, or 2 μg), and polyubiquitination was analyzed by immunoblotting using anti-Flag antibody. (B) An in vitro ubiquitination assay was performed using the protein pair GST-UCP ∆N /GST-UCP C95A (each 0.2 μg) and His-VHL (2 μg). His-VHL protein was pulled down with Ni-NTA agarose, and ubiquitinated forms were analyzed by immunoblotting using anti-Flag antibody. (C) HA-VHL and UCP WT or UCP mutants (UCP C95A , UCP C118A and UCP ∆N ) or various pairs of UCP mutant (UCP C95A /UCP ΔN , UCP ΔN /UCP C118A ) plasmids (total of 10 μg) were co-transfected into HEK-293T cells and treated with/without 10 μM MG132 for 12 h. At 48 h post-transfection, the cells were harvested and lysed. Changes in pVHL expression levels were detected by immunoblotting. (D) An in vitro ubiquitination assay was performed using wild-type UCP (0.2 μg) or UCP mutants (GST-UCP C118A and GST-UCP ∆N , each 0.2 μg) and His-UCP C95A (2 μg). His-UCP C95A protein was pulled down with Ni-NTA agarose, and ubiquitinated forms of His-UCP C95A were assessed by immunoblotting using anti-Flag antibody. (E) An in vitro ubiquitination assay was performed using wild-type UCP (0.2 μg), double-mutant UCP with K76R and K100R (His-UCP K76R/K100R, 0.2 μg), C118A-mutant UCP (His-UCP C118A, 0.2 μg) and GST-UCP C95A or GST-UCP CA (2 μg) at 37°C for 1 h. The GST-UCP C95A or GST-UCP CA protein was pulled down with GST agarose and analyzed by immunoblotting using anti-Flag antibody.

Techniques Used: In Vitro, Ubiquitin Assay, Mutagenesis, Transfection, Expressing

The N-terminal and Core Domains of UCP are Critical for Substrate Binding. (A) His-UCP WT (1 μg) and GST-UCP WT (1 μg) or truncated UCP mutants (UCP N-term , UCP Core , UCP C-term , UCP ∆N and UCP ∆C ) (1 μg) were incubated at 4°C for 2 h. His-UCP was then pulled down with Ni-NTA agarose at 4°C for 2 h, and the bound domains were detected by immunoblotting. (B) His-VHL (1 μg) and GST-UCP WT (1 μg) or truncated UCP mutants (1 μg) were incubated at 4°C for 2 h. His-UCP was then pulled down with Ni-NTA agarose, and the bound domains were detected by immunoblotting. (C) HA-VHL (5 μg) and GST-UCP WT (5 μg) or truncated UCP mutant plasmids (5 μg, each) were co-transfected into HEK-293T cells. GST-UCP proteins were pulled down with GST-resin, and then HA-VHL was detected by immunoblotting. (D) The plasmids Flag-UCP WT (5 μg) and GST-VHL WT or truncated VHL mutants (β, α, ∆ECEB) (5 μg, each) were co-transfected into HEK-293T cells. GST-VHL was pulled down with GST-agarose, and then interaction with UCP was detected by immunoblotting using anti-Flag antibody. (E, F) Schematic representation of UCP and pVHL. The functional domains of UCP and pVHL are delineated by three colored boxes (white, gray and black). The ability of the different domains to bind UCP and VHL is indicated; +; binding; -; no binding.
Figure Legend Snippet: The N-terminal and Core Domains of UCP are Critical for Substrate Binding. (A) His-UCP WT (1 μg) and GST-UCP WT (1 μg) or truncated UCP mutants (UCP N-term , UCP Core , UCP C-term , UCP ∆N and UCP ∆C ) (1 μg) were incubated at 4°C for 2 h. His-UCP was then pulled down with Ni-NTA agarose at 4°C for 2 h, and the bound domains were detected by immunoblotting. (B) His-VHL (1 μg) and GST-UCP WT (1 μg) or truncated UCP mutants (1 μg) were incubated at 4°C for 2 h. His-UCP was then pulled down with Ni-NTA agarose, and the bound domains were detected by immunoblotting. (C) HA-VHL (5 μg) and GST-UCP WT (5 μg) or truncated UCP mutant plasmids (5 μg, each) were co-transfected into HEK-293T cells. GST-UCP proteins were pulled down with GST-resin, and then HA-VHL was detected by immunoblotting. (D) The plasmids Flag-UCP WT (5 μg) and GST-VHL WT or truncated VHL mutants (β, α, ∆ECEB) (5 μg, each) were co-transfected into HEK-293T cells. GST-VHL was pulled down with GST-agarose, and then interaction with UCP was detected by immunoblotting using anti-Flag antibody. (E, F) Schematic representation of UCP and pVHL. The functional domains of UCP and pVHL are delineated by three colored boxes (white, gray and black). The ability of the different domains to bind UCP and VHL is indicated; +; binding; -; no binding.

Techniques Used: Binding Assay, Incubation, Mutagenesis, Transfection, Functional Assay

4) Product Images from "E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue"

Article Title: E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue

Journal: PLoS ONE

doi: 10.1371/journal.pone.0163710

The Residue Cys118 Plays a Key Role in the Autoubiquitination of UCP. (A) An autoubiquitination assay was performed using His-UCP WT and His-UCP C118A (0.2 μg each) along with wild-type ubiquitin (Ub WT ) at various time points. Ubiquitinated forms were detected by immunoblotting using anti-ubiquitin antibody. (B) An autoubiquitination assay was performed using His-UCP WT and His-UCP C118A (0.2 μg each) along with lysine-null ubiquitin (Ub ∆K ) at various time points. Ubiquitinated forms were detected by immunoblotting using anti-ubiquitin antibody. (C) In vitro ubiquitination assay was performed using His-UCP WT (0.2 μg) and GST-UCP C95A (2 μg) at various time points. GST-UCP C95A was pulled down using GST agarose and then polyubiquitination was separated by SDS-PAGE under denaturing or non-denaturing (without β-mercaptoethanol) condition. The polyubiquitin chains were detected by anti-Flag antibody. (D) Illustration of the expected reaction steps for polyubiquitination by UCP in the trans manner and the status of polyubiquitin chains on the substrate under different conditions. (E) An in vitro ubiquitination assay was performed using wild-type UCP (His-UCP WT ), double-mutant UCP with K78R and K100R (His-UCP K76R/K100R ), UCP-C118A mutant (His-UCP C118A ) (0.2 μg each) and GST-UCP C95A (2 μg) at 37°C for 1 h in reaction buffer. After the reaction, GST-UCP C95A was pulled down with GST agarose, and polyubiquitination was analyzed by immunoblotting under denaturing (+β-mercaptoethanol) or non-denaturing (-β-mercaptoethanol) conditions using anti-Flag antibody.
Figure Legend Snippet: The Residue Cys118 Plays a Key Role in the Autoubiquitination of UCP. (A) An autoubiquitination assay was performed using His-UCP WT and His-UCP C118A (0.2 μg each) along with wild-type ubiquitin (Ub WT ) at various time points. Ubiquitinated forms were detected by immunoblotting using anti-ubiquitin antibody. (B) An autoubiquitination assay was performed using His-UCP WT and His-UCP C118A (0.2 μg each) along with lysine-null ubiquitin (Ub ∆K ) at various time points. Ubiquitinated forms were detected by immunoblotting using anti-ubiquitin antibody. (C) In vitro ubiquitination assay was performed using His-UCP WT (0.2 μg) and GST-UCP C95A (2 μg) at various time points. GST-UCP C95A was pulled down using GST agarose and then polyubiquitination was separated by SDS-PAGE under denaturing or non-denaturing (without β-mercaptoethanol) condition. The polyubiquitin chains were detected by anti-Flag antibody. (D) Illustration of the expected reaction steps for polyubiquitination by UCP in the trans manner and the status of polyubiquitin chains on the substrate under different conditions. (E) An in vitro ubiquitination assay was performed using wild-type UCP (His-UCP WT ), double-mutant UCP with K78R and K100R (His-UCP K76R/K100R ), UCP-C118A mutant (His-UCP C118A ) (0.2 μg each) and GST-UCP C95A (2 μg) at 37°C for 1 h in reaction buffer. After the reaction, GST-UCP C95A was pulled down with GST agarose, and polyubiquitination was analyzed by immunoblotting under denaturing (+β-mercaptoethanol) or non-denaturing (-β-mercaptoethanol) conditions using anti-Flag antibody.

Techniques Used: In Vitro, Ubiquitin Assay, SDS Page, Mutagenesis

UCP Forms Polyubiquitin Chains on Specific Lysine Residues in its Substrate. (A) In vitro ubiquitination assays were performed using His-UCP WT as the enzyme and inactive UCP mutants containing lysine-to-arginine mutations as the substrates (GST-UCP-C95A, GST-UCP- C95A/K76R, GST-UCP-C95A/K100R, and GST-UCP-C95A/K76R,K100R) (2 μg). After the reaction, GST-UCP was pulled down with GST agarose at 4°C for 2 h. The ubiquitinated forms were visualized with anti-Flag antibody. (B) An in vitro ubiquitination assay was performed using His-UCP WT (0.2 μg) and wild-type and/or single-lysine pVHL mutants (VHL K159 , VHL K171 , VHL K196 and VHL ∆K ) (2 μg) at 37°C for 1 h. After incubation, pVHL was pulled down with GST agarose, and ubiquitinated forms were detected by immunoblotting using anti-Flag antibody. (C) Wild-type pVHL (5 μg), single-lysine pVHL (5 μg) mutant and HA-Ubiquitin plasmids (2 μg) were co-transfected into HEK-293T cells, which were then incubated with 10 μM MG132 for 12 h. After the cells were lysed, pVHL was pulled down with GST agarose, and ubiquitinated forms were detected by immunoblotting with anti-HA antibody.
Figure Legend Snippet: UCP Forms Polyubiquitin Chains on Specific Lysine Residues in its Substrate. (A) In vitro ubiquitination assays were performed using His-UCP WT as the enzyme and inactive UCP mutants containing lysine-to-arginine mutations as the substrates (GST-UCP-C95A, GST-UCP- C95A/K76R, GST-UCP-C95A/K100R, and GST-UCP-C95A/K76R,K100R) (2 μg). After the reaction, GST-UCP was pulled down with GST agarose at 4°C for 2 h. The ubiquitinated forms were visualized with anti-Flag antibody. (B) An in vitro ubiquitination assay was performed using His-UCP WT (0.2 μg) and wild-type and/or single-lysine pVHL mutants (VHL K159 , VHL K171 , VHL K196 and VHL ∆K ) (2 μg) at 37°C for 1 h. After incubation, pVHL was pulled down with GST agarose, and ubiquitinated forms were detected by immunoblotting using anti-Flag antibody. (C) Wild-type pVHL (5 μg), single-lysine pVHL (5 μg) mutant and HA-Ubiquitin plasmids (2 μg) were co-transfected into HEK-293T cells, which were then incubated with 10 μM MG132 for 12 h. After the cells were lysed, pVHL was pulled down with GST agarose, and ubiquitinated forms were detected by immunoblotting with anti-HA antibody.

Techniques Used: In Vitro, Ubiquitin Assay, Incubation, Mutagenesis, Transfection

Autoubiquitination of UCP Occurs in an E3-independent Manner. (A) An autoubiquitination assay was performed using UCP at various time points. GST-UCP (0.2 μg) was incubated at 37°C in the presence of E1 and Flag-ubiquitin, and autoubiquitination was visualized with anti-Flag antibody. (B) The catalytic ability of UncH5c proteins was analyzed in an in vitro autoubiquitination assay. GST-UbcH5c proteins (0.2 μg) and GST-UCP (0.2 μg) were incubated at 37°C for 40 min in the presence of E1 and Flag-ubiquitin, and the ubiquitinated proteins were detected by immunoblotting using anti-Flag antibody. (C) The lysine-specific linkage of UCP was defined using lysine-to-arginine ubiquitin mutants (K6R, K11R, K48R and K63R), single-lysine ubiquitin mutants (K6, K11, K48 and K63) and a lysine-null ubiquitin mutant (K-null). Autoubiquitination assays were performed using GST-UCP (0.2 μg) and wild type ubiquitin or ubiquitin mutants at 37°C for 1 h, and ubiquitinated proteins were detected by immunoblotting using anti-ubiquitin antibody. (D) GST-UCP (0.2 μg) and His-UCP C95A (2 μg) were incubated at 37°C for 1 h in the presence of E1 and Flag-ubiquitin. His-UCP C95A was then pulled down with Ni-NTA agarose, and His-UCP C95A polyubiquitination was detected by immunoblotting using anti-Flag antibody.
Figure Legend Snippet: Autoubiquitination of UCP Occurs in an E3-independent Manner. (A) An autoubiquitination assay was performed using UCP at various time points. GST-UCP (0.2 μg) was incubated at 37°C in the presence of E1 and Flag-ubiquitin, and autoubiquitination was visualized with anti-Flag antibody. (B) The catalytic ability of UncH5c proteins was analyzed in an in vitro autoubiquitination assay. GST-UbcH5c proteins (0.2 μg) and GST-UCP (0.2 μg) were incubated at 37°C for 40 min in the presence of E1 and Flag-ubiquitin, and the ubiquitinated proteins were detected by immunoblotting using anti-Flag antibody. (C) The lysine-specific linkage of UCP was defined using lysine-to-arginine ubiquitin mutants (K6R, K11R, K48R and K63R), single-lysine ubiquitin mutants (K6, K11, K48 and K63) and a lysine-null ubiquitin mutant (K-null). Autoubiquitination assays were performed using GST-UCP (0.2 μg) and wild type ubiquitin or ubiquitin mutants at 37°C for 1 h, and ubiquitinated proteins were detected by immunoblotting using anti-ubiquitin antibody. (D) GST-UCP (0.2 μg) and His-UCP C95A (2 μg) were incubated at 37°C for 1 h in the presence of E1 and Flag-ubiquitin. His-UCP C95A was then pulled down with Ni-NTA agarose, and His-UCP C95A polyubiquitination was detected by immunoblotting using anti-Flag antibody.

Techniques Used: Incubation, In Vitro, Mutagenesis

The UBC Domain of UCP can Forms Polyubiquitin Chains on the N-terminus. (A) The catalytic activity of UCP WT (0.2 μg) and truncated UCP mutants (UCP N-term , UCP Core , UCP C-term , UCP ∆N and UCP ∆C ) (0.2 μg) was assessed using an in vitro ubiquitination assay. The proteins were incubated at 37°C for 1 h in the presence of E1 and His-ubiquitin, and the ubiquitinated proteins were detected by immunoblotting using anti-His antibody. (B) The substrate region of UCP for autoubiquitination was analyzed in stable HeLa cell lines transfected with shRNA-control or shRNA-UCP. Different stable HeLa cell lines were co-transfected with plasmids encoding each truncated UCP mutant (5 μg) and HA-Ubiquitin (2 μg), treated with 10μM MG132 for 12 h and harvested at 48 h post-transfection. The cells were then lysed, and UCP was pulled down with GST-agarose. The ubiquitinated domains were subsequently detected by immunoblotting using anti-HA antibody. (C) GST-tagged UCP WT (0.2 μg) and truncated UCP mutants (UCP C-term and UCP ∆C , 0.2 μg) were mixed with His-VHL (2 μg) and subjected to an in vitro ubiquitination assays at 37°C for 1 h. pVHL was pulled down with Ni-NTA agarose, and ubiquitinated forms were detected by immunoblotting using anti-Flag antibody. (D) HA-VHL (5 μg) and GST-wild-type UCP or truncated UCP mutant plasmids (5 μg) were co-transfected into HEK-293T cells and treated with/without 10 μM MG132 for 12 h. Changes in the pVHL expression level were then detected by immunoblotting.
Figure Legend Snippet: The UBC Domain of UCP can Forms Polyubiquitin Chains on the N-terminus. (A) The catalytic activity of UCP WT (0.2 μg) and truncated UCP mutants (UCP N-term , UCP Core , UCP C-term , UCP ∆N and UCP ∆C ) (0.2 μg) was assessed using an in vitro ubiquitination assay. The proteins were incubated at 37°C for 1 h in the presence of E1 and His-ubiquitin, and the ubiquitinated proteins were detected by immunoblotting using anti-His antibody. (B) The substrate region of UCP for autoubiquitination was analyzed in stable HeLa cell lines transfected with shRNA-control or shRNA-UCP. Different stable HeLa cell lines were co-transfected with plasmids encoding each truncated UCP mutant (5 μg) and HA-Ubiquitin (2 μg), treated with 10μM MG132 for 12 h and harvested at 48 h post-transfection. The cells were then lysed, and UCP was pulled down with GST-agarose. The ubiquitinated domains were subsequently detected by immunoblotting using anti-HA antibody. (C) GST-tagged UCP WT (0.2 μg) and truncated UCP mutants (UCP C-term and UCP ∆C , 0.2 μg) were mixed with His-VHL (2 μg) and subjected to an in vitro ubiquitination assays at 37°C for 1 h. pVHL was pulled down with Ni-NTA agarose, and ubiquitinated forms were detected by immunoblotting using anti-Flag antibody. (D) HA-VHL (5 μg) and GST-wild-type UCP or truncated UCP mutant plasmids (5 μg) were co-transfected into HEK-293T cells and treated with/without 10 μM MG132 for 12 h. Changes in the pVHL expression level were then detected by immunoblotting.

Techniques Used: Activity Assay, In Vitro, Ubiquitin Assay, Incubation, Transfection, shRNA, Mutagenesis, Expressing

Intermolecular Active Cys118 Residues are Required for Autoubiquitination of UCP. (A) Autoubiquitination was assessed using GST-UCP C95A (1 μg) and GST-UCP ∆N (0.5, 1, or 2 μg), and polyubiquitination was analyzed by immunoblotting using anti-Flag antibody. (B) An in vitro ubiquitination assay was performed using the protein pair GST-UCP ∆N /GST-UCP C95A (each 0.2 μg) and His-VHL (2 μg). His-VHL protein was pulled down with Ni-NTA agarose, and ubiquitinated forms were analyzed by immunoblotting using anti-Flag antibody. (C) HA-VHL and UCP WT or UCP mutants (UCP C95A , UCP C118A and UCP ∆N ) or various pairs of UCP mutant (UCP C95A /UCP ΔN , UCP ΔN /UCP C118A ) plasmids (total of 10 μg) were co-transfected into HEK-293T cells and treated with/without 10 μM MG132 for 12 h. At 48 h post-transfection, the cells were harvested and lysed. Changes in pVHL expression levels were detected by immunoblotting. (D) An in vitro ubiquitination assay was performed using wild-type UCP (0.2 μg) or UCP mutants (GST-UCP C118A and GST-UCP ∆N , each 0.2 μg) and His-UCP C95A (2 μg). His-UCP C95A protein was pulled down with Ni-NTA agarose, and ubiquitinated forms of His-UCP C95A were assessed by immunoblotting using anti-Flag antibody. (E) An in vitro ubiquitination assay was performed using wild-type UCP (0.2 μg), double-mutant UCP with K76R and K100R (His-UCP K76R/K100R, 0.2 μg), C118A-mutant UCP (His-UCP C118A, 0.2 μg) and GST-UCP C95A or GST-UCP CA (2 μg) at 37°C for 1 h. The GST-UCP C95A or GST-UCP CA protein was pulled down with GST agarose and analyzed by immunoblotting using anti-Flag antibody.
Figure Legend Snippet: Intermolecular Active Cys118 Residues are Required for Autoubiquitination of UCP. (A) Autoubiquitination was assessed using GST-UCP C95A (1 μg) and GST-UCP ∆N (0.5, 1, or 2 μg), and polyubiquitination was analyzed by immunoblotting using anti-Flag antibody. (B) An in vitro ubiquitination assay was performed using the protein pair GST-UCP ∆N /GST-UCP C95A (each 0.2 μg) and His-VHL (2 μg). His-VHL protein was pulled down with Ni-NTA agarose, and ubiquitinated forms were analyzed by immunoblotting using anti-Flag antibody. (C) HA-VHL and UCP WT or UCP mutants (UCP C95A , UCP C118A and UCP ∆N ) or various pairs of UCP mutant (UCP C95A /UCP ΔN , UCP ΔN /UCP C118A ) plasmids (total of 10 μg) were co-transfected into HEK-293T cells and treated with/without 10 μM MG132 for 12 h. At 48 h post-transfection, the cells were harvested and lysed. Changes in pVHL expression levels were detected by immunoblotting. (D) An in vitro ubiquitination assay was performed using wild-type UCP (0.2 μg) or UCP mutants (GST-UCP C118A and GST-UCP ∆N , each 0.2 μg) and His-UCP C95A (2 μg). His-UCP C95A protein was pulled down with Ni-NTA agarose, and ubiquitinated forms of His-UCP C95A were assessed by immunoblotting using anti-Flag antibody. (E) An in vitro ubiquitination assay was performed using wild-type UCP (0.2 μg), double-mutant UCP with K76R and K100R (His-UCP K76R/K100R, 0.2 μg), C118A-mutant UCP (His-UCP C118A, 0.2 μg) and GST-UCP C95A or GST-UCP CA (2 μg) at 37°C for 1 h. The GST-UCP C95A or GST-UCP CA protein was pulled down with GST agarose and analyzed by immunoblotting using anti-Flag antibody.

Techniques Used: In Vitro, Ubiquitin Assay, Mutagenesis, Transfection, Expressing

The N-terminal and Core Domains of UCP are Critical for Substrate Binding. (A) His-UCP WT (1 μg) and GST-UCP WT (1 μg) or truncated UCP mutants (UCP N-term , UCP Core , UCP C-term , UCP ∆N and UCP ∆C ) (1 μg) were incubated at 4°C for 2 h. His-UCP was then pulled down with Ni-NTA agarose at 4°C for 2 h, and the bound domains were detected by immunoblotting. (B) His-VHL (1 μg) and GST-UCP WT (1 μg) or truncated UCP mutants (1 μg) were incubated at 4°C for 2 h. His-UCP was then pulled down with Ni-NTA agarose, and the bound domains were detected by immunoblotting. (C) HA-VHL (5 μg) and GST-UCP WT (5 μg) or truncated UCP mutant plasmids (5 μg, each) were co-transfected into HEK-293T cells. GST-UCP proteins were pulled down with GST-resin, and then HA-VHL was detected by immunoblotting. (D) The plasmids Flag-UCP WT (5 μg) and GST-VHL WT or truncated VHL mutants (β, α, ∆ECEB) (5 μg, each) were co-transfected into HEK-293T cells. GST-VHL was pulled down with GST-agarose, and then interaction with UCP was detected by immunoblotting using anti-Flag antibody. (E, F) Schematic representation of UCP and pVHL. The functional domains of UCP and pVHL are delineated by three colored boxes (white, gray and black). The ability of the different domains to bind UCP and VHL is indicated; +; binding; -; no binding.
Figure Legend Snippet: The N-terminal and Core Domains of UCP are Critical for Substrate Binding. (A) His-UCP WT (1 μg) and GST-UCP WT (1 μg) or truncated UCP mutants (UCP N-term , UCP Core , UCP C-term , UCP ∆N and UCP ∆C ) (1 μg) were incubated at 4°C for 2 h. His-UCP was then pulled down with Ni-NTA agarose at 4°C for 2 h, and the bound domains were detected by immunoblotting. (B) His-VHL (1 μg) and GST-UCP WT (1 μg) or truncated UCP mutants (1 μg) were incubated at 4°C for 2 h. His-UCP was then pulled down with Ni-NTA agarose, and the bound domains were detected by immunoblotting. (C) HA-VHL (5 μg) and GST-UCP WT (5 μg) or truncated UCP mutant plasmids (5 μg, each) were co-transfected into HEK-293T cells. GST-UCP proteins were pulled down with GST-resin, and then HA-VHL was detected by immunoblotting. (D) The plasmids Flag-UCP WT (5 μg) and GST-VHL WT or truncated VHL mutants (β, α, ∆ECEB) (5 μg, each) were co-transfected into HEK-293T cells. GST-VHL was pulled down with GST-agarose, and then interaction with UCP was detected by immunoblotting using anti-Flag antibody. (E, F) Schematic representation of UCP and pVHL. The functional domains of UCP and pVHL are delineated by three colored boxes (white, gray and black). The ability of the different domains to bind UCP and VHL is indicated; +; binding; -; no binding.

Techniques Used: Binding Assay, Incubation, Mutagenesis, Transfection, Functional Assay

5) Product Images from "ORF73 of Herpesvirus Saimiri, a Viral Homolog of Kaposi's Sarcoma-Associated Herpesvirus, Modulates the Two Cellular Tumor Suppressor Proteins p53 and pRb"

Article Title: ORF73 of Herpesvirus Saimiri, a Viral Homolog of Kaposi's Sarcoma-Associated Herpesvirus, Modulates the Two Cellular Tumor Suppressor Proteins p53 and pRb

Journal: Journal of Virology

doi: 10.1128/JVI.78.19.10336-10347.2004

LANA preferentially binds to the tetramerization domain of p53 in vitro. (A) Full-length p53 and the p53 transactivation domain (TR), DNA-binding domain (DBD), and tetramerization domain (TET) fused to GST were expressed in E. coli DH5α cells and purified from sonicated cell lysate with glutathione-Sepharose 4B beads (Amersham-Pharmacia). (B) Luciferase and LANA protein were in vitro transcribed and translated in the presence of [ 35 S]methionine and cysteine and incubated with equal amounts of purified p53 constructs for 4 h at 4°C. Pulled-down, in vitro-translated product was resolved on an 8% Tris-glycine gel.
Figure Legend Snippet: LANA preferentially binds to the tetramerization domain of p53 in vitro. (A) Full-length p53 and the p53 transactivation domain (TR), DNA-binding domain (DBD), and tetramerization domain (TET) fused to GST were expressed in E. coli DH5α cells and purified from sonicated cell lysate with glutathione-Sepharose 4B beads (Amersham-Pharmacia). (B) Luciferase and LANA protein were in vitro transcribed and translated in the presence of [ 35 S]methionine and cysteine and incubated with equal amounts of purified p53 constructs for 4 h at 4°C. Pulled-down, in vitro-translated product was resolved on an 8% Tris-glycine gel.

Techniques Used: In Vitro, Binding Assay, Purification, Sonication, Luciferase, Incubation, Construct

C-terminal 400 amino acids of KSHV LANA bind p53 in vitro. (A) LANA deletion constructs spanning residues 1 to 435, 1 to 756, 1 to 950, and 752 to 1162 were in vitro transcribed and translated in the presence of [ 35 S]methionine and then incubated with equal amounts of either GST or GST-p53 glutathione-Sepharose beads for 4 h at 4°C. (B) Retained radiolabeled protein was resolved on 7% Tris-glycine gels. Bands were quantified by phophorimager analysis (Amersham Pharmacia, Inc., Uppsala, Sweden).
Figure Legend Snippet: C-terminal 400 amino acids of KSHV LANA bind p53 in vitro. (A) LANA deletion constructs spanning residues 1 to 435, 1 to 756, 1 to 950, and 752 to 1162 were in vitro transcribed and translated in the presence of [ 35 S]methionine and then incubated with equal amounts of either GST or GST-p53 glutathione-Sepharose beads for 4 h at 4°C. (B) Retained radiolabeled protein was resolved on 7% Tris-glycine gels. Bands were quantified by phophorimager analysis (Amersham Pharmacia, Inc., Uppsala, Sweden).

Techniques Used: In Vitro, Construct, Incubation

pRb coimmunoprecipitates with LANA and HVS ORF73 in vivo and in vitro. (A) Full-length LANA and deletion constructs were in vitro translated in the presence of [ 35 S]methionine (T N T-T 7 rabbit reticulate lysate) and incubated with either GST or GST-pRb glutathione-Sepharose beads for 4 h at 4°C. Immunoprecipitated proteins were resolved on a 7% Tris-glycine-SDS gel. (B) We transfected 20 million 293T cells with: lane 1, vector alone; lane 2, pRb alone; lane 3, LANA alone; lane 4, LANA and pRb together; lane 5, HVS C488 ORF73 alone; lane 6, HVS C488 ORF73 and pRb together; lane 7, HVS A11 ORF73 alone; or lane 8, HVS A11 ORF73 and pRb together. Cells were harvested 20 h later and lysed with RIPA buffer. Protein complexes were immunoprecipitated with anti-Myc ascites fluid and resolved on a 7% Tris-glycine gel.
Figure Legend Snippet: pRb coimmunoprecipitates with LANA and HVS ORF73 in vivo and in vitro. (A) Full-length LANA and deletion constructs were in vitro translated in the presence of [ 35 S]methionine (T N T-T 7 rabbit reticulate lysate) and incubated with either GST or GST-pRb glutathione-Sepharose beads for 4 h at 4°C. Immunoprecipitated proteins were resolved on a 7% Tris-glycine-SDS gel. (B) We transfected 20 million 293T cells with: lane 1, vector alone; lane 2, pRb alone; lane 3, LANA alone; lane 4, LANA and pRb together; lane 5, HVS C488 ORF73 alone; lane 6, HVS C488 ORF73 and pRb together; lane 7, HVS A11 ORF73 alone; or lane 8, HVS A11 ORF73 and pRb together. Cells were harvested 20 h later and lysed with RIPA buffer. Protein complexes were immunoprecipitated with anti-Myc ascites fluid and resolved on a 7% Tris-glycine gel.

Techniques Used: In Vivo, In Vitro, Construct, Incubation, Immunoprecipitation, SDS-Gel, Transfection, Plasmid Preparation

KSHV LANA and HVS ORF73 bind to p53 in vitro. (A) GST (left panel) and GST-p53 (middle and right panels) were purified from bacterial cell lysate with glutathione-Sepharose 4B beads (Amersham Pharmacia, Inc., Piscataway, N.J.). Uninduced lysate, induced lysate, and pulled-down proteins were resolved on a 10% Tris-glycine-SDS gels. GST-p53 protein expression and purification were also confirmed by Western blot analysis (right panel), as described in Materials and Methods. M, markers; U, uninduced lysate; I, induced lysate; WB, Western blot. (B) Side by side comparison of the functional domains and distinct sequence motifs shared by KSHV LANA (top) and HVS ORF73 (bottom). NLS, nuclear localization signal; LIM, the LIM domain which mediates protein-protein interactions via cysteine-mediated coordination of zinc ions. (C) GST and GST-p53 pulldowns of [ 35 S]methionine-labeled, in vitro-translated luciferase, in vitro-translated KSHV LANA, in vitro-translated ORF73 from HVS strain A11, and in vitro-translated ORF73 from HVS strain C488.
Figure Legend Snippet: KSHV LANA and HVS ORF73 bind to p53 in vitro. (A) GST (left panel) and GST-p53 (middle and right panels) were purified from bacterial cell lysate with glutathione-Sepharose 4B beads (Amersham Pharmacia, Inc., Piscataway, N.J.). Uninduced lysate, induced lysate, and pulled-down proteins were resolved on a 10% Tris-glycine-SDS gels. GST-p53 protein expression and purification were also confirmed by Western blot analysis (right panel), as described in Materials and Methods. M, markers; U, uninduced lysate; I, induced lysate; WB, Western blot. (B) Side by side comparison of the functional domains and distinct sequence motifs shared by KSHV LANA (top) and HVS ORF73 (bottom). NLS, nuclear localization signal; LIM, the LIM domain which mediates protein-protein interactions via cysteine-mediated coordination of zinc ions. (C) GST and GST-p53 pulldowns of [ 35 S]methionine-labeled, in vitro-translated luciferase, in vitro-translated KSHV LANA, in vitro-translated ORF73 from HVS strain A11, and in vitro-translated ORF73 from HVS strain C488.

Techniques Used: In Vitro, Purification, Expressing, Western Blot, Functional Assay, Sequencing, Labeling, Luciferase

p53 coimmunoprecipitates in vivo with KSHV LANA and HVS ORF73. (A) We transfected 20 million 293T cells with: lane 1, empty vector alone (mock); lane 2, p53 expression vector alone; lane 3, Myc-tagged LANA alone; lane 4, Myc-tagged LANA and p53 expression vector together; lane 5, Myc-tagged HVS C488 ORF73 alone; lane 6, Myc-tagged HVS C488 ORF73 and p53 expression vector together; lane 7, Myc-tagged HVS A11 ORF73 alone; or lane 8, Myc-tagged HVS A11 ORF73 and p53 expression vector together. Cells were harvested 20 h later, lysed with RIPA buffer, and incubated with anti-Myc mouse ascites fluid overnight, which targeted the tagged LANA/ORF73 proteins. Antibody-bound LANA/ORF73 proteins and associated protein complexes were immunoprecipitated the next day with a protein A-protein G-Sepharose bead slurry (Amersham Pharmacia, Inc., Piscataway, N.J.) and resolved on 12% Tris-glycine gels, which were blotted with either anti-Myc antibody (top panels) or anti-human p53 antibody (bottom panels) (Santa Cruz Biotechnologies, Inc.). The mouse immunoglobulin G heavy chain used to immunoprecipitate Myc-tagged proteins migrates slightly below the p53 protein and is indicated HC. p53 is marked by white arrows. (B) An inverse pulldown strategy was also employed by immunoprecipitating LANA and ORF73 with bacterially prepared GST-p53; 10 million 293T cells were transfected with either KSHV LANA (left panels) or HVS A11 ORF73 (right panels). Cells were lysed with RIPA buffer and incubated with either glutathione-Sepharose-bound GST or GST-p53. Washed beads were resuspended in 2× SDS buffer. Input, GST-pulled-down and GST-p53-pulled-down fractions were resolved on an 8% Tris-glycine gel. LANA and ORF73 were detected by blotting with an anti-Myc antibody (α-Myc WB), and p53 was detected by blotting with anti-p53 antibody (α-p53 WB).
Figure Legend Snippet: p53 coimmunoprecipitates in vivo with KSHV LANA and HVS ORF73. (A) We transfected 20 million 293T cells with: lane 1, empty vector alone (mock); lane 2, p53 expression vector alone; lane 3, Myc-tagged LANA alone; lane 4, Myc-tagged LANA and p53 expression vector together; lane 5, Myc-tagged HVS C488 ORF73 alone; lane 6, Myc-tagged HVS C488 ORF73 and p53 expression vector together; lane 7, Myc-tagged HVS A11 ORF73 alone; or lane 8, Myc-tagged HVS A11 ORF73 and p53 expression vector together. Cells were harvested 20 h later, lysed with RIPA buffer, and incubated with anti-Myc mouse ascites fluid overnight, which targeted the tagged LANA/ORF73 proteins. Antibody-bound LANA/ORF73 proteins and associated protein complexes were immunoprecipitated the next day with a protein A-protein G-Sepharose bead slurry (Amersham Pharmacia, Inc., Piscataway, N.J.) and resolved on 12% Tris-glycine gels, which were blotted with either anti-Myc antibody (top panels) or anti-human p53 antibody (bottom panels) (Santa Cruz Biotechnologies, Inc.). The mouse immunoglobulin G heavy chain used to immunoprecipitate Myc-tagged proteins migrates slightly below the p53 protein and is indicated HC. p53 is marked by white arrows. (B) An inverse pulldown strategy was also employed by immunoprecipitating LANA and ORF73 with bacterially prepared GST-p53; 10 million 293T cells were transfected with either KSHV LANA (left panels) or HVS A11 ORF73 (right panels). Cells were lysed with RIPA buffer and incubated with either glutathione-Sepharose-bound GST or GST-p53. Washed beads were resuspended in 2× SDS buffer. Input, GST-pulled-down and GST-p53-pulled-down fractions were resolved on an 8% Tris-glycine gel. LANA and ORF73 were detected by blotting with an anti-Myc antibody (α-Myc WB), and p53 was detected by blotting with anti-p53 antibody (α-p53 WB).

Techniques Used: In Vivo, Transfection, Plasmid Preparation, Expressing, Incubation, Immunoprecipitation, Western Blot

6) Product Images from "Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap"

Article Title: Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2016.02119

Interaction of DDX3 with PAdV-3 and HAdV-5 pVIII. (A) Coomassie blue staining of purified protein. Purified GST.DDX3 protein was separated by 10% SDS-PAGE and stained with 0.25 Coomassie blue stain. (B) GST-pull down assay. Purified GSTor GST.DDX3 fusion protein immobilized on Glutathione-Sepharose 4B beads, incubated individually with in vitro translated [ 35 S] methionine labeled PAdV-3 pVIII or HAdV-5 pVIII, separated by 10% SDS-PAGE and detected by autoradiography. (C) Co-immunoprecipitation. Radio labeled in vitro transcribed and translated HAdV5 pVIII or PAdV-3 pVIII was incubated with in vitro transcribed and translated unlabeled DDX3 protein. Proteins were immunoprecipitated with either anti-DDX3 serum or rabbit pre immune sera, separated by 10% SDS-PAGE and auto radio-graphed. Immunoprecipitation (IP).
Figure Legend Snippet: Interaction of DDX3 with PAdV-3 and HAdV-5 pVIII. (A) Coomassie blue staining of purified protein. Purified GST.DDX3 protein was separated by 10% SDS-PAGE and stained with 0.25 Coomassie blue stain. (B) GST-pull down assay. Purified GSTor GST.DDX3 fusion protein immobilized on Glutathione-Sepharose 4B beads, incubated individually with in vitro translated [ 35 S] methionine labeled PAdV-3 pVIII or HAdV-5 pVIII, separated by 10% SDS-PAGE and detected by autoradiography. (C) Co-immunoprecipitation. Radio labeled in vitro transcribed and translated HAdV5 pVIII or PAdV-3 pVIII was incubated with in vitro transcribed and translated unlabeled DDX3 protein. Proteins were immunoprecipitated with either anti-DDX3 serum or rabbit pre immune sera, separated by 10% SDS-PAGE and auto radio-graphed. Immunoprecipitation (IP).

Techniques Used: Staining, Purification, SDS Page, Pull Down Assay, Incubation, In Vitro, Labeling, Autoradiography, Immunoprecipitation

Effect of pVIII on capped mRNA translation. (A). In vitro . The TNT ® T7 luciferase DNA (Promega) (i) was transcribed in vitro in the absence (uncapped) or presence (capped) of 40 mM Ribo m7GpppG cap analog (Promega) using RiboMAX RNA production system-T7 (Promega). The in vitro synthesized capped and uncapped luciferase mRNAs (ii) were translated in the supernatant collected after centrifugation of mixture of Flexi Rabbit Reticulo Lysate (Promega) incubated with Glutathione sepharose beads preloaded with GST.VIII or GST protein alone. The level of luciferase activity was measured using a luciferase kit (Promega) on a Luminometer (Turner Designs, Inc.). The results are shown as relative luciferase activity (iii). Error bars indicate SE of means for separate experiments. The relative luciferase intensity is determined based on GST compared to GST.pVIII. (B) In vivo . 293T cells were transfected with plasmid DNAs (2 μg of pcDNA3-RLuc-POLIRES-FLuc (i) and either 4 μg of pEY.pVIII or 4 μg of pEYFPN1). At 36 h post transfection, Firefly luciferase (FLuc) and Renilla reniformis luciferase (RLuc) activities were measured in a luminometer by using a dual luciferase assay kit (Promega) as per the company’s procedure. Expression of EYFP was used to normalize the transfection efficiency. The results are shown as relative luciferase activity (iii). The level of cytoplasmic RLuc-POLIRES-FLuc mRNA both in EY.pVIII and EYFP expressing plasmid transfected cells was quantified by RT-PCR (ii). Error bars indicate SE of means for three separate experiments. ∗ statistically significant.
Figure Legend Snippet: Effect of pVIII on capped mRNA translation. (A). In vitro . The TNT ® T7 luciferase DNA (Promega) (i) was transcribed in vitro in the absence (uncapped) or presence (capped) of 40 mM Ribo m7GpppG cap analog (Promega) using RiboMAX RNA production system-T7 (Promega). The in vitro synthesized capped and uncapped luciferase mRNAs (ii) were translated in the supernatant collected after centrifugation of mixture of Flexi Rabbit Reticulo Lysate (Promega) incubated with Glutathione sepharose beads preloaded with GST.VIII or GST protein alone. The level of luciferase activity was measured using a luciferase kit (Promega) on a Luminometer (Turner Designs, Inc.). The results are shown as relative luciferase activity (iii). Error bars indicate SE of means for separate experiments. The relative luciferase intensity is determined based on GST compared to GST.pVIII. (B) In vivo . 293T cells were transfected with plasmid DNAs (2 μg of pcDNA3-RLuc-POLIRES-FLuc (i) and either 4 μg of pEY.pVIII or 4 μg of pEYFPN1). At 36 h post transfection, Firefly luciferase (FLuc) and Renilla reniformis luciferase (RLuc) activities were measured in a luminometer by using a dual luciferase assay kit (Promega) as per the company’s procedure. Expression of EYFP was used to normalize the transfection efficiency. The results are shown as relative luciferase activity (iii). The level of cytoplasmic RLuc-POLIRES-FLuc mRNA both in EY.pVIII and EYFP expressing plasmid transfected cells was quantified by RT-PCR (ii). Error bars indicate SE of means for three separate experiments. ∗ statistically significant.

Techniques Used: In Vitro, Luciferase, Synthesized, Centrifugation, Incubation, Activity Assay, In Vivo, Transfection, Plasmid Preparation, Expressing, Reverse Transcription Polymerase Chain Reaction

m7GTP-sepharose binding assay. (A) The supernatant of the lysates of the cells collected at 36 h post BAdV-3 infection of MDBK cells (mock or BAdV-3) or transfection of 293T cells with plasmid DNAs (pEY.pVIII or pEYFPN1) were incubated with m7GTP sepharose cap affinity beads. After washing, the bound proteins were analyzed by Western blot using indicated protein specific antibodies and IRDye 800 conjugated goat anti-mouse IgG or Alexa Flour 680 goat anti-rabbit IgG as secondary antibody. The intensity of the bands of the Western blot in all cases was analyzed by Odyssey Software v2.1. The relative amount of proteins in BAdV-3 infected or pEY.VIII transfected cell lysates that are retained in the 7-methyl GTP resins as compared to mock infected or pEYFPN1 transfected cells, respectively (i.e., considering the amount of protein in mock infected or pEYFPN1 transfected cell lysates that are retained in the m7GTP resins as 100%) is plotted. Error bars indicate SE of means for three separate experiments. Proteins from the lysates of BAdV-3 infected or transfected cells were separated by 10% SDS-PAGE and probed in Western blot using anti-pVIII serum. (B) Proteins from the lysates of mock infected or BAdV-3 infected MDBK cells collected at 36 h post infection were separated by 10% SDS-PAGE and analyzed by Western blot using protein specific antibody and anti-rabbit IRDye 800 conjugated goat anti-mouse IgG (Li-COR biosciences) or Alexa Flour 680 goat anti-rabbit IgG as secondary antibody. β-actin was used as a loading control.
Figure Legend Snippet: m7GTP-sepharose binding assay. (A) The supernatant of the lysates of the cells collected at 36 h post BAdV-3 infection of MDBK cells (mock or BAdV-3) or transfection of 293T cells with plasmid DNAs (pEY.pVIII or pEYFPN1) were incubated with m7GTP sepharose cap affinity beads. After washing, the bound proteins were analyzed by Western blot using indicated protein specific antibodies and IRDye 800 conjugated goat anti-mouse IgG or Alexa Flour 680 goat anti-rabbit IgG as secondary antibody. The intensity of the bands of the Western blot in all cases was analyzed by Odyssey Software v2.1. The relative amount of proteins in BAdV-3 infected or pEY.VIII transfected cell lysates that are retained in the 7-methyl GTP resins as compared to mock infected or pEYFPN1 transfected cells, respectively (i.e., considering the amount of protein in mock infected or pEYFPN1 transfected cell lysates that are retained in the m7GTP resins as 100%) is plotted. Error bars indicate SE of means for three separate experiments. Proteins from the lysates of BAdV-3 infected or transfected cells were separated by 10% SDS-PAGE and probed in Western blot using anti-pVIII serum. (B) Proteins from the lysates of mock infected or BAdV-3 infected MDBK cells collected at 36 h post infection were separated by 10% SDS-PAGE and analyzed by Western blot using protein specific antibody and anti-rabbit IRDye 800 conjugated goat anti-mouse IgG (Li-COR biosciences) or Alexa Flour 680 goat anti-rabbit IgG as secondary antibody. β-actin was used as a loading control.

Techniques Used: Binding Assay, Infection, Transfection, Plasmid Preparation, Incubation, Western Blot, Software, SDS Page

Interaction of DDX3 with BAdV-3 pVIII. (A) Glutathione S-transferase (GST) pull down assay. Purified GST or GST.pVIII fusion protein immobilized on Glutathione-Sepharose 4B beads, incubated with in vitro translated [ 35 S] methionine labeled HA tagged DDX3 were separated by 10% SDS-PAGE and detected by autoradiography. (B,C) Co-immunoprecipitation in transfected cells. Proteins from the lysates of cells co-transfected with either pHA.DX3 and pEY.pVIII or pHA.DX3 and pEYFPN1 were immunoprecipitated with anti-pVIII serum (B) or anti-HA MAb (C) , separated by 10% SDS-PAGE and transferred to nitrocellulose membrane. The separated proteins were probed in Western blot using anti-HA MAb (B) or anti-pVIII serum (C) . (D) Co-immunoprecipitation in BAdV-3 infected cells. Proteins from the lysates of mock or BAdV-3 infected Madin-Darby Bovine Kidney (MDBK) cells were immunoprecipitated with anti-pVIII serum, separated by 10% SDS-PAGE, transferred to nitrocellulose membrane and probed in Western blot using anti-DDX3 MAb. Immunoprecipitation (IP). WB (Western blot). Ctl (Control) . (E–G) Confocal microscopy. MDBK cells mock infected (panels a and f) or infected with BAdV-3 (panels d and g1–g4) VERO cells untransfected (panel b) or transfected with indicated plasmid (panels c, e, and h1–h4) DNA, were fixed 36 h post-infection/transfection. The subcellular localization of DDX3 (panels a–c, g2, and h2) protein was visualized by indirect immunofluorescence (panels a–c, g2, h2) using anti-DDX3 MAb and fluorescein conjugated goat anti-mouse IgG-FITC (panels a and g2), anti-DDX3 MAb and Cy3 conjugated goat anti-mouse (pane b) secondary antibody, anti-HA MAb and Cy3 conjugated goat anti-mouse secondary antibody (panel c and h2). The subcellular localization of pVIII (panels d, e, f, g1, and h1) was visualized by direct fluorescence (panels e and h1) or indirect immunofluorescence using anti-pVIII serum and Cy3 conjugated goat anti-rabbit secondary antibody (panels d, f, and g1). Nuclei were stained with DAPI in each panel. A merge of the images is shown. Enlargement of panel g4 and h4 is shown, arrows in white shows few of the colocalization of pVIII and DDX3.
Figure Legend Snippet: Interaction of DDX3 with BAdV-3 pVIII. (A) Glutathione S-transferase (GST) pull down assay. Purified GST or GST.pVIII fusion protein immobilized on Glutathione-Sepharose 4B beads, incubated with in vitro translated [ 35 S] methionine labeled HA tagged DDX3 were separated by 10% SDS-PAGE and detected by autoradiography. (B,C) Co-immunoprecipitation in transfected cells. Proteins from the lysates of cells co-transfected with either pHA.DX3 and pEY.pVIII or pHA.DX3 and pEYFPN1 were immunoprecipitated with anti-pVIII serum (B) or anti-HA MAb (C) , separated by 10% SDS-PAGE and transferred to nitrocellulose membrane. The separated proteins were probed in Western blot using anti-HA MAb (B) or anti-pVIII serum (C) . (D) Co-immunoprecipitation in BAdV-3 infected cells. Proteins from the lysates of mock or BAdV-3 infected Madin-Darby Bovine Kidney (MDBK) cells were immunoprecipitated with anti-pVIII serum, separated by 10% SDS-PAGE, transferred to nitrocellulose membrane and probed in Western blot using anti-DDX3 MAb. Immunoprecipitation (IP). WB (Western blot). Ctl (Control) . (E–G) Confocal microscopy. MDBK cells mock infected (panels a and f) or infected with BAdV-3 (panels d and g1–g4) VERO cells untransfected (panel b) or transfected with indicated plasmid (panels c, e, and h1–h4) DNA, were fixed 36 h post-infection/transfection. The subcellular localization of DDX3 (panels a–c, g2, and h2) protein was visualized by indirect immunofluorescence (panels a–c, g2, h2) using anti-DDX3 MAb and fluorescein conjugated goat anti-mouse IgG-FITC (panels a and g2), anti-DDX3 MAb and Cy3 conjugated goat anti-mouse (pane b) secondary antibody, anti-HA MAb and Cy3 conjugated goat anti-mouse secondary antibody (panel c and h2). The subcellular localization of pVIII (panels d, e, f, g1, and h1) was visualized by direct fluorescence (panels e and h1) or indirect immunofluorescence using anti-pVIII serum and Cy3 conjugated goat anti-rabbit secondary antibody (panels d, f, and g1). Nuclei were stained with DAPI in each panel. A merge of the images is shown. Enlargement of panel g4 and h4 is shown, arrows in white shows few of the colocalization of pVIII and DDX3.

Techniques Used: Pull Down Assay, Purification, Incubation, In Vitro, Labeling, SDS Page, Autoradiography, Immunoprecipitation, Transfection, Western Blot, Infection, CTL Assay, Confocal Microscopy, Plasmid Preparation, Immunofluorescence, Fluorescence, Staining

7) Product Images from "Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap"

Article Title: Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2016.02119

Interaction of DDX3 with PAdV-3 and HAdV-5 pVIII. (A) Coomassie blue staining of purified protein. Purified GST.DDX3 protein was separated by 10% SDS-PAGE and stained with 0.25 Coomassie blue stain. (B) GST-pull down assay. Purified GSTor GST.DDX3 fusion protein immobilized on Glutathione-Sepharose 4B beads, incubated individually with in vitro translated [ 35 S] methionine labeled PAdV-3 pVIII or HAdV-5 pVIII, separated by 10% SDS-PAGE and detected by autoradiography. (C) Co-immunoprecipitation. Radio labeled in vitro transcribed and translated HAdV5 pVIII or PAdV-3 pVIII was incubated with in vitro transcribed and translated unlabeled DDX3 protein. Proteins were immunoprecipitated with either anti-DDX3 serum or rabbit pre immune sera, separated by 10% SDS-PAGE and auto radio-graphed. Immunoprecipitation (IP).
Figure Legend Snippet: Interaction of DDX3 with PAdV-3 and HAdV-5 pVIII. (A) Coomassie blue staining of purified protein. Purified GST.DDX3 protein was separated by 10% SDS-PAGE and stained with 0.25 Coomassie blue stain. (B) GST-pull down assay. Purified GSTor GST.DDX3 fusion protein immobilized on Glutathione-Sepharose 4B beads, incubated individually with in vitro translated [ 35 S] methionine labeled PAdV-3 pVIII or HAdV-5 pVIII, separated by 10% SDS-PAGE and detected by autoradiography. (C) Co-immunoprecipitation. Radio labeled in vitro transcribed and translated HAdV5 pVIII or PAdV-3 pVIII was incubated with in vitro transcribed and translated unlabeled DDX3 protein. Proteins were immunoprecipitated with either anti-DDX3 serum or rabbit pre immune sera, separated by 10% SDS-PAGE and auto radio-graphed. Immunoprecipitation (IP).

Techniques Used: Staining, Purification, SDS Page, Pull Down Assay, Incubation, In Vitro, Labeling, Autoradiography, Immunoprecipitation

Effect of pVIII on capped mRNA translation. (A). In vitro . The TNT ® T7 luciferase DNA (Promega) (i) was transcribed in vitro in the absence (uncapped) or presence (capped) of 40 mM Ribo m7GpppG cap analog (Promega) using RiboMAX RNA production system-T7 (Promega). The in vitro synthesized capped and uncapped luciferase mRNAs (ii) were translated in the supernatant collected after centrifugation of mixture of Flexi Rabbit Reticulo Lysate (Promega) incubated with Glutathione sepharose beads preloaded with GST.VIII or GST protein alone. The level of luciferase activity was measured using a luciferase kit (Promega) on a Luminometer (Turner Designs, Inc.). The results are shown as relative luciferase activity (iii). Error bars indicate SE of means for separate experiments. The relative luciferase intensity is determined based on GST compared to GST.pVIII. (B) In vivo . 293T cells were transfected with plasmid DNAs (2 μg of pcDNA3-RLuc-POLIRES-FLuc (i) and either 4 μg of pEY.pVIII or 4 μg of pEYFPN1). At 36 h post transfection, Firefly luciferase (FLuc) and Renilla reniformis luciferase (RLuc) activities were measured in a luminometer by using a dual luciferase assay kit (Promega) as per the company’s procedure. Expression of EYFP was used to normalize the transfection efficiency. The results are shown as relative luciferase activity (iii). The level of cytoplasmic RLuc-POLIRES-FLuc mRNA both in EY.pVIII and EYFP expressing plasmid transfected cells was quantified by RT-PCR (ii). Error bars indicate SE of means for three separate experiments. ∗ statistically significant.
Figure Legend Snippet: Effect of pVIII on capped mRNA translation. (A). In vitro . The TNT ® T7 luciferase DNA (Promega) (i) was transcribed in vitro in the absence (uncapped) or presence (capped) of 40 mM Ribo m7GpppG cap analog (Promega) using RiboMAX RNA production system-T7 (Promega). The in vitro synthesized capped and uncapped luciferase mRNAs (ii) were translated in the supernatant collected after centrifugation of mixture of Flexi Rabbit Reticulo Lysate (Promega) incubated with Glutathione sepharose beads preloaded with GST.VIII or GST protein alone. The level of luciferase activity was measured using a luciferase kit (Promega) on a Luminometer (Turner Designs, Inc.). The results are shown as relative luciferase activity (iii). Error bars indicate SE of means for separate experiments. The relative luciferase intensity is determined based on GST compared to GST.pVIII. (B) In vivo . 293T cells were transfected with plasmid DNAs (2 μg of pcDNA3-RLuc-POLIRES-FLuc (i) and either 4 μg of pEY.pVIII or 4 μg of pEYFPN1). At 36 h post transfection, Firefly luciferase (FLuc) and Renilla reniformis luciferase (RLuc) activities were measured in a luminometer by using a dual luciferase assay kit (Promega) as per the company’s procedure. Expression of EYFP was used to normalize the transfection efficiency. The results are shown as relative luciferase activity (iii). The level of cytoplasmic RLuc-POLIRES-FLuc mRNA both in EY.pVIII and EYFP expressing plasmid transfected cells was quantified by RT-PCR (ii). Error bars indicate SE of means for three separate experiments. ∗ statistically significant.

Techniques Used: In Vitro, Luciferase, Synthesized, Centrifugation, Incubation, Activity Assay, In Vivo, Transfection, Plasmid Preparation, Expressing, Reverse Transcription Polymerase Chain Reaction

m7GTP-sepharose binding assay. (A) The supernatant of the lysates of the cells collected at 36 h post BAdV-3 infection of MDBK cells (mock or BAdV-3) or transfection of 293T cells with plasmid DNAs (pEY.pVIII or pEYFPN1) were incubated with m7GTP sepharose cap affinity beads. After washing, the bound proteins were analyzed by Western blot using indicated protein specific antibodies and IRDye 800 conjugated goat anti-mouse IgG or Alexa Flour 680 goat anti-rabbit IgG as secondary antibody. The intensity of the bands of the Western blot in all cases was analyzed by Odyssey Software v2.1. The relative amount of proteins in BAdV-3 infected or pEY.VIII transfected cell lysates that are retained in the 7-methyl GTP resins as compared to mock infected or pEYFPN1 transfected cells, respectively (i.e., considering the amount of protein in mock infected or pEYFPN1 transfected cell lysates that are retained in the m7GTP resins as 100%) is plotted. Error bars indicate SE of means for three separate experiments. Proteins from the lysates of BAdV-3 infected or transfected cells were separated by 10% SDS-PAGE and probed in Western blot using anti-pVIII serum. (B) Proteins from the lysates of mock infected or BAdV-3 infected MDBK cells collected at 36 h post infection were separated by 10% SDS-PAGE and analyzed by Western blot using protein specific antibody and anti-rabbit IRDye 800 conjugated goat anti-mouse IgG (Li-COR biosciences) or Alexa Flour 680 goat anti-rabbit IgG as secondary antibody. β-actin was used as a loading control.
Figure Legend Snippet: m7GTP-sepharose binding assay. (A) The supernatant of the lysates of the cells collected at 36 h post BAdV-3 infection of MDBK cells (mock or BAdV-3) or transfection of 293T cells with plasmid DNAs (pEY.pVIII or pEYFPN1) were incubated with m7GTP sepharose cap affinity beads. After washing, the bound proteins were analyzed by Western blot using indicated protein specific antibodies and IRDye 800 conjugated goat anti-mouse IgG or Alexa Flour 680 goat anti-rabbit IgG as secondary antibody. The intensity of the bands of the Western blot in all cases was analyzed by Odyssey Software v2.1. The relative amount of proteins in BAdV-3 infected or pEY.VIII transfected cell lysates that are retained in the 7-methyl GTP resins as compared to mock infected or pEYFPN1 transfected cells, respectively (i.e., considering the amount of protein in mock infected or pEYFPN1 transfected cell lysates that are retained in the m7GTP resins as 100%) is plotted. Error bars indicate SE of means for three separate experiments. Proteins from the lysates of BAdV-3 infected or transfected cells were separated by 10% SDS-PAGE and probed in Western blot using anti-pVIII serum. (B) Proteins from the lysates of mock infected or BAdV-3 infected MDBK cells collected at 36 h post infection were separated by 10% SDS-PAGE and analyzed by Western blot using protein specific antibody and anti-rabbit IRDye 800 conjugated goat anti-mouse IgG (Li-COR biosciences) or Alexa Flour 680 goat anti-rabbit IgG as secondary antibody. β-actin was used as a loading control.

Techniques Used: Binding Assay, Infection, Transfection, Plasmid Preparation, Incubation, Western Blot, Software, SDS Page

Interaction of DDX3 with BAdV-3 pVIII. (A) Glutathione S-transferase (GST) pull down assay. Purified GST or GST.pVIII fusion protein immobilized on Glutathione-Sepharose 4B beads, incubated with in vitro translated [ 35 S] methionine labeled HA tagged DDX3 were separated by 10% SDS-PAGE and detected by autoradiography. (B,C) Co-immunoprecipitation in transfected cells. Proteins from the lysates of cells co-transfected with either pHA.DX3 and pEY.pVIII or pHA.DX3 and pEYFPN1 were immunoprecipitated with anti-pVIII serum (B) or anti-HA MAb (C) , separated by 10% SDS-PAGE and transferred to nitrocellulose membrane. The separated proteins were probed in Western blot using anti-HA MAb (B) or anti-pVIII serum (C) . (D) Co-immunoprecipitation in BAdV-3 infected cells. Proteins from the lysates of mock or BAdV-3 infected Madin-Darby Bovine Kidney (MDBK) cells were immunoprecipitated with anti-pVIII serum, separated by 10% SDS-PAGE, transferred to nitrocellulose membrane and probed in Western blot using anti-DDX3 MAb. Immunoprecipitation (IP). WB (Western blot). Ctl (Control) . (E–G) Confocal microscopy. MDBK cells mock infected (panels a and f) or infected with BAdV-3 (panels d and g1–g4) VERO cells untransfected (panel b) or transfected with indicated plasmid (panels c, e, and h1–h4) DNA, were fixed 36 h post-infection/transfection. The subcellular localization of DDX3 (panels a–c, g2, and h2) protein was visualized by indirect immunofluorescence (panels a–c, g2, h2) using anti-DDX3 MAb and fluorescein conjugated goat anti-mouse IgG-FITC (panels a and g2), anti-DDX3 MAb and Cy3 conjugated goat anti-mouse (pane b) secondary antibody, anti-HA MAb and Cy3 conjugated goat anti-mouse secondary antibody (panel c and h2). The subcellular localization of pVIII (panels d, e, f, g1, and h1) was visualized by direct fluorescence (panels e and h1) or indirect immunofluorescence using anti-pVIII serum and Cy3 conjugated goat anti-rabbit secondary antibody (panels d, f, and g1). Nuclei were stained with DAPI in each panel. A merge of the images is shown. Enlargement of panel g4 and h4 is shown, arrows in white shows few of the colocalization of pVIII and DDX3.
Figure Legend Snippet: Interaction of DDX3 with BAdV-3 pVIII. (A) Glutathione S-transferase (GST) pull down assay. Purified GST or GST.pVIII fusion protein immobilized on Glutathione-Sepharose 4B beads, incubated with in vitro translated [ 35 S] methionine labeled HA tagged DDX3 were separated by 10% SDS-PAGE and detected by autoradiography. (B,C) Co-immunoprecipitation in transfected cells. Proteins from the lysates of cells co-transfected with either pHA.DX3 and pEY.pVIII or pHA.DX3 and pEYFPN1 were immunoprecipitated with anti-pVIII serum (B) or anti-HA MAb (C) , separated by 10% SDS-PAGE and transferred to nitrocellulose membrane. The separated proteins were probed in Western blot using anti-HA MAb (B) or anti-pVIII serum (C) . (D) Co-immunoprecipitation in BAdV-3 infected cells. Proteins from the lysates of mock or BAdV-3 infected Madin-Darby Bovine Kidney (MDBK) cells were immunoprecipitated with anti-pVIII serum, separated by 10% SDS-PAGE, transferred to nitrocellulose membrane and probed in Western blot using anti-DDX3 MAb. Immunoprecipitation (IP). WB (Western blot). Ctl (Control) . (E–G) Confocal microscopy. MDBK cells mock infected (panels a and f) or infected with BAdV-3 (panels d and g1–g4) VERO cells untransfected (panel b) or transfected with indicated plasmid (panels c, e, and h1–h4) DNA, were fixed 36 h post-infection/transfection. The subcellular localization of DDX3 (panels a–c, g2, and h2) protein was visualized by indirect immunofluorescence (panels a–c, g2, h2) using anti-DDX3 MAb and fluorescein conjugated goat anti-mouse IgG-FITC (panels a and g2), anti-DDX3 MAb and Cy3 conjugated goat anti-mouse (pane b) secondary antibody, anti-HA MAb and Cy3 conjugated goat anti-mouse secondary antibody (panel c and h2). The subcellular localization of pVIII (panels d, e, f, g1, and h1) was visualized by direct fluorescence (panels e and h1) or indirect immunofluorescence using anti-pVIII serum and Cy3 conjugated goat anti-rabbit secondary antibody (panels d, f, and g1). Nuclei were stained with DAPI in each panel. A merge of the images is shown. Enlargement of panel g4 and h4 is shown, arrows in white shows few of the colocalization of pVIII and DDX3.

Techniques Used: Pull Down Assay, Purification, Incubation, In Vitro, Labeling, SDS Page, Autoradiography, Immunoprecipitation, Transfection, Western Blot, Infection, CTL Assay, Confocal Microscopy, Plasmid Preparation, Immunofluorescence, Fluorescence, Staining

8) Product Images from "The Sec34/Sec35p complex, a Ypt1p effector required for retrograde intra-Golgi trafficking, interacts with Golgi SNAREs and COPI vesicle coat proteins"

Article Title: The Sec34/Sec35p complex, a Ypt1p effector required for retrograde intra-Golgi trafficking, interacts with Golgi SNAREs and COPI vesicle coat proteins

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200111081

GST-tagged TFI1, TFI2, and TFI3 interact with both Sed5p and COPI proteins. GST-tagged proteins were expressed in the cells of appropriate gene deletion strains, in which the GST-chimera was the only source of TFI1, TFI2, or TFI3 proteins. A membrane fraction was obtained after centrifugation at 150,000 g for 1 h at 4°C. Extracted membrane proteins (2 mg) were incubated with 50 μl prewashed glutathione–Sepharose beads. Each eluate was loaded on the 10% SDS-PAGE and analyzed by immunoblot with α-Sed5p and α-coatomer sera. The Coomassie blue staining of the samples presents on the left panel of the Fig. 1 B.
Figure Legend Snippet: GST-tagged TFI1, TFI2, and TFI3 interact with both Sed5p and COPI proteins. GST-tagged proteins were expressed in the cells of appropriate gene deletion strains, in which the GST-chimera was the only source of TFI1, TFI2, or TFI3 proteins. A membrane fraction was obtained after centrifugation at 150,000 g for 1 h at 4°C. Extracted membrane proteins (2 mg) were incubated with 50 μl prewashed glutathione–Sepharose beads. Each eluate was loaded on the 10% SDS-PAGE and analyzed by immunoblot with α-Sed5p and α-coatomer sera. The Coomassie blue staining of the samples presents on the left panel of the Fig. 1 B.

Techniques Used: Centrifugation, Incubation, SDS Page, Staining

Sec34p interacts with retrograde Golgi SNAREs and with COPI. GST-Sec34p was expressed in a Δsec34 yeast strain. Affinity chromatography on glutathione–Sepharose beads was employed to purify GST-Sec34p and associated proteins from a P100 fraction (Total) that was solubilized in CHN buffer (20 mM Hepes, pH 7.4, 1% CHAPS, 0.15 M NaCl). The beads were eluted with 10 mM glutathione (Eluate). As a control, the GST protein was expressed and purified from the membrane fraction of sec34/ Δ sec34 strain. The GST-Sec34p– and Sec34-associated proteins were identified by immunoblotting with antibodies to A, SNARE proteins and Sec35p; (B) anti-COPI, Sec21p, or Sec13p. (C) Coomassie blue staining of the GST and GST-Sec34p eluates.
Figure Legend Snippet: Sec34p interacts with retrograde Golgi SNAREs and with COPI. GST-Sec34p was expressed in a Δsec34 yeast strain. Affinity chromatography on glutathione–Sepharose beads was employed to purify GST-Sec34p and associated proteins from a P100 fraction (Total) that was solubilized in CHN buffer (20 mM Hepes, pH 7.4, 1% CHAPS, 0.15 M NaCl). The beads were eluted with 10 mM glutathione (Eluate). As a control, the GST protein was expressed and purified from the membrane fraction of sec34/ Δ sec34 strain. The GST-Sec34p– and Sec34-associated proteins were identified by immunoblotting with antibodies to A, SNARE proteins and Sec35p; (B) anti-COPI, Sec21p, or Sec13p. (C) Coomassie blue staining of the GST and GST-Sec34p eluates.

Techniques Used: Affinity Chromatography, Purification, Staining

The Sec34/35 complex is an effector of the Ypt1 protein . GST-Ypt1 or GST-Ypt6p (1 μg each), were preloaded with GDP or GTP as indicated, and bound to glutathione–Sepharose beads. Beads were incubated with 0.2 μg of purified Sec34/35 complex in 0.2 ml of binding buffer (40 mM Hepes pH 7.0, 150 mM KoAc, 2 mM MgOAc, 5% glycerol, 1 mM DTT) for 3 h at 4°C. Beads were washed with binding buffer and proteins were eluted with the sample buffer. Eluted proteins and 25% of the unbound material were loaded on 10% SDS-PAGE and immunoblotted with affinity-purified antibodies to GST, Sec34p, and Sec35p.
Figure Legend Snippet: The Sec34/35 complex is an effector of the Ypt1 protein . GST-Ypt1 or GST-Ypt6p (1 μg each), were preloaded with GDP or GTP as indicated, and bound to glutathione–Sepharose beads. Beads were incubated with 0.2 μg of purified Sec34/35 complex in 0.2 ml of binding buffer (40 mM Hepes pH 7.0, 150 mM KoAc, 2 mM MgOAc, 5% glycerol, 1 mM DTT) for 3 h at 4°C. Beads were washed with binding buffer and proteins were eluted with the sample buffer. Eluted proteins and 25% of the unbound material were loaded on 10% SDS-PAGE and immunoblotted with affinity-purified antibodies to GST, Sec34p, and Sec35p.

Techniques Used: Incubation, Purification, Binding Assay, SDS Page, Affinity Purification

Sec35p-TAP complex purified from yeast cytosol. (A) Sec35p-TAP is associated with four other proteins. Proteins eluted from calmodulin-agarose beads were separated by gel electrophoresis and stained with Coomassie blue. The indicated bands were identified by mass spectroscopy of tryptic fragments. The band labeled with double asterisks represents a proteolytic fragment of Tfi3p. No readable spectra were obtained from the minor bands labeled with asterisks. (B) GST-tagged TFI1, TFI2, and TFI3 interact with both Sec34p and Sec35p. The GST-tagged proteins were expressed in cells of appropriate gene deletion strains, in which the GST-chimera was the only source of TFI1, TFI2, or TFI3 protein. Membrane fractions were obtained after centrifugation at 150,000 g , 1 h, 4°C. Extracted membrane proteins (2 mg) were incubated with 50 μl prewashed glutathione–Sepharose beads. The eluates were loaded on the 10% SDS-PAGE and then analyzed by immunoblot with α Sec34p and α Sec35p (left), or stained with Coomassie blue (right).
Figure Legend Snippet: Sec35p-TAP complex purified from yeast cytosol. (A) Sec35p-TAP is associated with four other proteins. Proteins eluted from calmodulin-agarose beads were separated by gel electrophoresis and stained with Coomassie blue. The indicated bands were identified by mass spectroscopy of tryptic fragments. The band labeled with double asterisks represents a proteolytic fragment of Tfi3p. No readable spectra were obtained from the minor bands labeled with asterisks. (B) GST-tagged TFI1, TFI2, and TFI3 interact with both Sec34p and Sec35p. The GST-tagged proteins were expressed in cells of appropriate gene deletion strains, in which the GST-chimera was the only source of TFI1, TFI2, or TFI3 protein. Membrane fractions were obtained after centrifugation at 150,000 g , 1 h, 4°C. Extracted membrane proteins (2 mg) were incubated with 50 μl prewashed glutathione–Sepharose beads. The eluates were loaded on the 10% SDS-PAGE and then analyzed by immunoblot with α Sec34p and α Sec35p (left), or stained with Coomassie blue (right).

Techniques Used: Purification, Nucleic Acid Electrophoresis, Staining, Mass Spectrometry, Labeling, Centrifugation, Incubation, SDS Page

Sec34p and Sed5p interact genetically and physically. (A) Coimmunoprecipitations of putative partners of the Sec34/35 complex. A P100 membrane fraction (Total) from RSY1157 was solubilized in 20 mM Hepes-KOH, pH 7.4, with 0.1 M NaCl and 1% CHAPS. The extract was incubated with protein A Sepharose beads, to which different primary antibodies had been crosslinked. Rabbit IgGs were used as a control for unspecific binding. After washing of the beads, specifically bound proteins were eluted, run on 11%SDS-PAGE, and analyzed by immunoblotting. Two representative experiments performed under identical conditions are shown. Membranes (3% of total) were loaded on the first lane. Approximately 15% of Sec34p and 40% of Sec35p were recovered in the corresponding IP. Approximately 0.2% of Sed5p was specifically coprecipitated in both Sec34 and Sec35 IP's. (B) The Sec34/Sec35 complex interacts with Sed5p in vitro. Purified GST, GST-Sed5p, or GST-Sso1p (5 μg each) were mixed with 0.2 μg of purified Sec34/35 complex in 0.5 ml binding buffer, incubated for 3 h at 4°C, and centrifuged at 20,000 g for 10 min. The supernatant (0.45 ml) was incubated for 1 h at 4°C with 20 μl glutathione–Sepharose beads in the same buffer. Beads were washed and bound proteins were eluted with 10 mM glutathione, separated by 10% SDS-PAGE, and immunoblotted with affinity-purified antibodies to Sec34p and Sec35p. (C) Physical in vitro interaction of Sec34p with Sed5p. Purified proteins (as in B) were bound to glutathione–Sepharose beads, mixed with 5 μg of His 6 -Sec34p in 0.5 ml of binding buffer and incubated for 3h at 4°C with rotation. Beads were washed and bound proteins were eluted with 10 mM glutathione, separated by 11% SDS-PAGE and stained with Coomassie blue. Approximately 10% of His 6 -Sec34p were recovered with GST-Sed5p bound to glutathione–Sepharose beads under conditions used. (D) The sed5–1 and sec34–2 alleles display a synthetic lethal interaction. Diploid strains resulting from the mating of GWY234 ( sed5–1 ) with GWY95 ( sec34–2 ) (top) or GWY235 ( sed5–1 ) with GWY93 ( sec35–1 ) (bottom) were sporulated, tetrad dissected, and incubated on YPD plates at 30°C for 3 d. Eight representative tetrads for each dissection are shown.
Figure Legend Snippet: Sec34p and Sed5p interact genetically and physically. (A) Coimmunoprecipitations of putative partners of the Sec34/35 complex. A P100 membrane fraction (Total) from RSY1157 was solubilized in 20 mM Hepes-KOH, pH 7.4, with 0.1 M NaCl and 1% CHAPS. The extract was incubated with protein A Sepharose beads, to which different primary antibodies had been crosslinked. Rabbit IgGs were used as a control for unspecific binding. After washing of the beads, specifically bound proteins were eluted, run on 11%SDS-PAGE, and analyzed by immunoblotting. Two representative experiments performed under identical conditions are shown. Membranes (3% of total) were loaded on the first lane. Approximately 15% of Sec34p and 40% of Sec35p were recovered in the corresponding IP. Approximately 0.2% of Sed5p was specifically coprecipitated in both Sec34 and Sec35 IP's. (B) The Sec34/Sec35 complex interacts with Sed5p in vitro. Purified GST, GST-Sed5p, or GST-Sso1p (5 μg each) were mixed with 0.2 μg of purified Sec34/35 complex in 0.5 ml binding buffer, incubated for 3 h at 4°C, and centrifuged at 20,000 g for 10 min. The supernatant (0.45 ml) was incubated for 1 h at 4°C with 20 μl glutathione–Sepharose beads in the same buffer. Beads were washed and bound proteins were eluted with 10 mM glutathione, separated by 10% SDS-PAGE, and immunoblotted with affinity-purified antibodies to Sec34p and Sec35p. (C) Physical in vitro interaction of Sec34p with Sed5p. Purified proteins (as in B) were bound to glutathione–Sepharose beads, mixed with 5 μg of His 6 -Sec34p in 0.5 ml of binding buffer and incubated for 3h at 4°C with rotation. Beads were washed and bound proteins were eluted with 10 mM glutathione, separated by 11% SDS-PAGE and stained with Coomassie blue. Approximately 10% of His 6 -Sec34p were recovered with GST-Sed5p bound to glutathione–Sepharose beads under conditions used. (D) The sed5–1 and sec34–2 alleles display a synthetic lethal interaction. Diploid strains resulting from the mating of GWY234 ( sed5–1 ) with GWY95 ( sec34–2 ) (top) or GWY235 ( sed5–1 ) with GWY93 ( sec35–1 ) (bottom) were sporulated, tetrad dissected, and incubated on YPD plates at 30°C for 3 d. Eight representative tetrads for each dissection are shown.

Techniques Used: Incubation, Binding Assay, SDS Page, In Vitro, Purification, Affinity Purification, Staining, Dissection

9) Product Images from "RARα2 and PML-RAR similarities in the control of basal and retinoic acid induced myeloid maturation of acute myeloid leukemia cells"

Article Title: RARα2 and PML-RAR similarities in the control of basal and retinoic acid induced myeloid maturation of acute myeloid leukemia cells

Journal: Oncotarget

doi: 10.18632/oncotarget.10556

Functional and physical interactions between RARα2 and RARα1 or PML-RAR A . GST pull-down: the GST-tagged recombinant protein, GST-RARα2 and GST were used. The two recombinant proteins conjugated to Glutathione-Sepharose beads were incubated with extracts of COS-7 cells transfected with pcDNA3-RARα1 and treated with vehicle or ATRA (1 μM) for 4 hours. GST pull-down precipitates were blotted on nitro-cellulose filters, hybridized with an anti-RARα [RP alpha (F)] (left panel) and subsequently with an anti-GST antibody (right panel). The blot was not stripped between the two hybridizations. Input: cell extracts (10 μg of protein) representing 10% of the total amount of protein were subjected to Western blot analysis with the above anti-RARα antibody. B . GST pull-down: the GST-tagged recombinant proteins, GST-RARα1 and GST-RARα1DEF were used. The two recombinant proteins conjugated to Glutathione-Sepharose beads were incubated with extracts of COS-7 cells transfected with pHA-RARα2 as well as the negative controls, pHA-SNAIL plasmid and pcDNA3 plasmid containing the HA tag ( pHA ). Transfected cells were treated with vehicle or ATRA as in (A). As an internal control of the experiment, we performed a pull-down assay with the GST protein coupled to Glutathione-Sepharose beads on extracts of cells transfected with pHA-RARα2 . GST pull-down precipitates were subjected to Western blot analysis with anti-HA (upper panels) and anti-GST antibodies (lower panels). C . Far-Western: COS-7 cells were transfected with a pcDNA3 plasmid containing a haemoagglutinin (HA) tagged RAR a 2 cDNA ( pHA-RARα2 ). Cell extracts were precipitated with agarose beads conjugated with an anti-HA monoclonal antibody. The immuno-precipitates were subjected to Far-Western analysis using the following GST-tagged RARα1 recombinant proteins: GST-RARα1 = full-length RARα1; GST-RARα1ABC = RARα1 ABC regions; GST-RARα1DEF = RARα1 DEF regions; GST-RARα1DEFD(408-416) = RARα1 DEF regions lacking the H12 helix. Input: cell extracts (10 μg of protein) representing 10% of the total amount of protein used for the immune-precipitations were subjected to Western blot analysis with an anti-HA antibody. Each line shows cropped lanes of the same gel, hence the results can be compared across the lanes, as they were obtained with the same exposure time. D . Immunoprecipitations (IP): COS-7 cells were transfected with pcDNA3-RARα1 and pcDNA3-RARα2 alone or in combination. Sixteen hours following transfection, cells were treated with vehicle or ATRA (1 μM) for 4 hours. The indicated extracts were immuno-precipitated with anti- RARα2 antibodies and subjected to Western blot analysis with a different anti-RARα antibody [RP alpha (F)]. The two leftmost lanes represent controls of RARα1 transfected cells directly submitted to Western blot analysis without immuno-precipitation. Equivalent amounts of protein extracts were used to immuno-precipitate RARα2, as indicated by the levels of RARα2 [Ab25alpha2(A2) antibody] and RARα1 [Ab10alpha1(A1)antibody]in the extracts (input). Each line shows cropped lanes of the same gel, hence the results can be compared across the lanes, as they were obtained with the same exposure time. E . Immuno-precipitation (IP): COS-7 cells were co-transfected with wild-type (WT) RARα2 or WT RARα1 and RARα1 mutants and subjected to immune-precipitation and Western blot analysis as in (D). F . Immuno-precipitation (IP): COS-7 cells were co-transfected with wild-type (WT) RARα2 or WT PML-RAR and derived mutant. The extracts of transfected cells were treated and subjected to co-immuno-precipitation studies as in (D and E). Lanes 5,6, 11 and 12 represent controls of PML-RAR and PML-RAR-S873A transfected cells directly submitted to Western blot analysis without immuno-precipitation.
Figure Legend Snippet: Functional and physical interactions between RARα2 and RARα1 or PML-RAR A . GST pull-down: the GST-tagged recombinant protein, GST-RARα2 and GST were used. The two recombinant proteins conjugated to Glutathione-Sepharose beads were incubated with extracts of COS-7 cells transfected with pcDNA3-RARα1 and treated with vehicle or ATRA (1 μM) for 4 hours. GST pull-down precipitates were blotted on nitro-cellulose filters, hybridized with an anti-RARα [RP alpha (F)] (left panel) and subsequently with an anti-GST antibody (right panel). The blot was not stripped between the two hybridizations. Input: cell extracts (10 μg of protein) representing 10% of the total amount of protein were subjected to Western blot analysis with the above anti-RARα antibody. B . GST pull-down: the GST-tagged recombinant proteins, GST-RARα1 and GST-RARα1DEF were used. The two recombinant proteins conjugated to Glutathione-Sepharose beads were incubated with extracts of COS-7 cells transfected with pHA-RARα2 as well as the negative controls, pHA-SNAIL plasmid and pcDNA3 plasmid containing the HA tag ( pHA ). Transfected cells were treated with vehicle or ATRA as in (A). As an internal control of the experiment, we performed a pull-down assay with the GST protein coupled to Glutathione-Sepharose beads on extracts of cells transfected with pHA-RARα2 . GST pull-down precipitates were subjected to Western blot analysis with anti-HA (upper panels) and anti-GST antibodies (lower panels). C . Far-Western: COS-7 cells were transfected with a pcDNA3 plasmid containing a haemoagglutinin (HA) tagged RAR a 2 cDNA ( pHA-RARα2 ). Cell extracts were precipitated with agarose beads conjugated with an anti-HA monoclonal antibody. The immuno-precipitates were subjected to Far-Western analysis using the following GST-tagged RARα1 recombinant proteins: GST-RARα1 = full-length RARα1; GST-RARα1ABC = RARα1 ABC regions; GST-RARα1DEF = RARα1 DEF regions; GST-RARα1DEFD(408-416) = RARα1 DEF regions lacking the H12 helix. Input: cell extracts (10 μg of protein) representing 10% of the total amount of protein used for the immune-precipitations were subjected to Western blot analysis with an anti-HA antibody. Each line shows cropped lanes of the same gel, hence the results can be compared across the lanes, as they were obtained with the same exposure time. D . Immunoprecipitations (IP): COS-7 cells were transfected with pcDNA3-RARα1 and pcDNA3-RARα2 alone or in combination. Sixteen hours following transfection, cells were treated with vehicle or ATRA (1 μM) for 4 hours. The indicated extracts were immuno-precipitated with anti- RARα2 antibodies and subjected to Western blot analysis with a different anti-RARα antibody [RP alpha (F)]. The two leftmost lanes represent controls of RARα1 transfected cells directly submitted to Western blot analysis without immuno-precipitation. Equivalent amounts of protein extracts were used to immuno-precipitate RARα2, as indicated by the levels of RARα2 [Ab25alpha2(A2) antibody] and RARα1 [Ab10alpha1(A1)antibody]in the extracts (input). Each line shows cropped lanes of the same gel, hence the results can be compared across the lanes, as they were obtained with the same exposure time. E . Immuno-precipitation (IP): COS-7 cells were co-transfected with wild-type (WT) RARα2 or WT RARα1 and RARα1 mutants and subjected to immune-precipitation and Western blot analysis as in (D). F . Immuno-precipitation (IP): COS-7 cells were co-transfected with wild-type (WT) RARα2 or WT PML-RAR and derived mutant. The extracts of transfected cells were treated and subjected to co-immuno-precipitation studies as in (D and E). Lanes 5,6, 11 and 12 represent controls of PML-RAR and PML-RAR-S873A transfected cells directly submitted to Western blot analysis without immuno-precipitation.

Techniques Used: Functional Assay, Recombinant, Incubation, Transfection, Western Blot, Plasmid Preparation, Pull Down Assay, Immunoprecipitation, Derivative Assay, Mutagenesis

PML-RAR, RARα2 and RARα1 knock-down in COS-7 and NB4 cells A . COS-7 cells were transiently transfected with the pcDNA3, pcDNA3-RARα2 , pcDNA3-RARα1 and pSG5-PML-RAR plasmids in the presence of the indicated shRNA-containing retroviral vectors and corresponding void vector ( VOID ). Sixteen hours following transfection, cell extracts were subjected to Western blot analysis using an anti-RARα antibody [RP alpha (F)]. Actin is used as a loading control. ALLsh = shRNA targeting RARα1 (RARα.v1 and RARα.v3 mRNAs), RARα2 (RARα.v2 mRNA) and RARα4 (RARα.v4 mRNA); RA1sh = shRNA targeting RARα1; RA2sh = shRNA targeting RARα2; PMRsh = shRNA targeting PML-RAR; SCRsh = scramble shRNA (negative control). The (-) symbol represents extracts from COS-7 cell transfected in the absence of any shRNA. B . The indicated NB4 cell populations stably infected with shRNAs targeting PML-RAR ( PMRsh-NB4 ), RARα1 ( RA1sh-NB4 ), RARα2 ( RA2sh-NB4 ) or scramble shRNA ( SCRsh-NB4 ) as well parental NB4 cells ( NB4 ) were grown under standard conditions for 48 hours. Cell extracts were subjected to Western blot analysis using the same anti-RARα antibody as in ( A ). Actin is used as a loading control. C . Upper (WB = Western Blots): Extracts from the indicated COS-7 cells transfected with RARα1 and RARα2 expressing plasmids as well as the indicated NB4 cells [see (B)], were subjected to Western blot analysis with anti-RARα2 [Ab25alpha2(A2)] and β-actin (loading control) antibodies. SCRsh-NB4 = cell treated with vehicle (DMSO) for 24 hours; SCRsh-NB4+ATRA = cell treated with ATRA (1 μM) for 24 hours. Kazumi cells extracts are used as a control for RARα2 expression, as they contain high levels of the protein. Lower (IP = immunoprecipitations): Extracts from the indicated NB4 cell populations and parental NB4 cells [see (B)], were immuno-precipitated with an anti-RARα2 antibody [Ab25alpha2(A2)] coupled to Protein G sepharose beads. The immuno-precipitates were subjected to Western blot analysis with a different anti-RARα antibody [RP alpha (F)]. Equivalent amounts of protein extracts were used to immuno-precipitate RARα2, as indicated by the levels of actin in the extracts before addition of the anti-RARα2 antibody (input). D . Extracts from the indicated NB4 cell populations [see (B)] were immuno-precipitated with an anti-RARα1 antibody [Ab10alpha1(A1)] or mouse immunoglobulin G (IgG) coupled to Protein G Sepharose beads or Protein G Sepharose beads alone (-). The immuno-precipitates were subjected to Western blot analysis with a different anti-RARα antibody [RP alpha (F)]. The actin loading control of the immuno-precipitation experiment is shown (input). The calculated molecular weight (M.W.) of each protein is indicated on the left of each blot.
Figure Legend Snippet: PML-RAR, RARα2 and RARα1 knock-down in COS-7 and NB4 cells A . COS-7 cells were transiently transfected with the pcDNA3, pcDNA3-RARα2 , pcDNA3-RARα1 and pSG5-PML-RAR plasmids in the presence of the indicated shRNA-containing retroviral vectors and corresponding void vector ( VOID ). Sixteen hours following transfection, cell extracts were subjected to Western blot analysis using an anti-RARα antibody [RP alpha (F)]. Actin is used as a loading control. ALLsh = shRNA targeting RARα1 (RARα.v1 and RARα.v3 mRNAs), RARα2 (RARα.v2 mRNA) and RARα4 (RARα.v4 mRNA); RA1sh = shRNA targeting RARα1; RA2sh = shRNA targeting RARα2; PMRsh = shRNA targeting PML-RAR; SCRsh = scramble shRNA (negative control). The (-) symbol represents extracts from COS-7 cell transfected in the absence of any shRNA. B . The indicated NB4 cell populations stably infected with shRNAs targeting PML-RAR ( PMRsh-NB4 ), RARα1 ( RA1sh-NB4 ), RARα2 ( RA2sh-NB4 ) or scramble shRNA ( SCRsh-NB4 ) as well parental NB4 cells ( NB4 ) were grown under standard conditions for 48 hours. Cell extracts were subjected to Western blot analysis using the same anti-RARα antibody as in ( A ). Actin is used as a loading control. C . Upper (WB = Western Blots): Extracts from the indicated COS-7 cells transfected with RARα1 and RARα2 expressing plasmids as well as the indicated NB4 cells [see (B)], were subjected to Western blot analysis with anti-RARα2 [Ab25alpha2(A2)] and β-actin (loading control) antibodies. SCRsh-NB4 = cell treated with vehicle (DMSO) for 24 hours; SCRsh-NB4+ATRA = cell treated with ATRA (1 μM) for 24 hours. Kazumi cells extracts are used as a control for RARα2 expression, as they contain high levels of the protein. Lower (IP = immunoprecipitations): Extracts from the indicated NB4 cell populations and parental NB4 cells [see (B)], were immuno-precipitated with an anti-RARα2 antibody [Ab25alpha2(A2)] coupled to Protein G sepharose beads. The immuno-precipitates were subjected to Western blot analysis with a different anti-RARα antibody [RP alpha (F)]. Equivalent amounts of protein extracts were used to immuno-precipitate RARα2, as indicated by the levels of actin in the extracts before addition of the anti-RARα2 antibody (input). D . Extracts from the indicated NB4 cell populations [see (B)] were immuno-precipitated with an anti-RARα1 antibody [Ab10alpha1(A1)] or mouse immunoglobulin G (IgG) coupled to Protein G Sepharose beads or Protein G Sepharose beads alone (-). The immuno-precipitates were subjected to Western blot analysis with a different anti-RARα antibody [RP alpha (F)]. The actin loading control of the immuno-precipitation experiment is shown (input). The calculated molecular weight (M.W.) of each protein is indicated on the left of each blot.

Techniques Used: Transfection, shRNA, Plasmid Preparation, Western Blot, Negative Control, Stable Transfection, Infection, Expressing, Immunoprecipitation, Molecular Weight

Effects of RARα2 over-expression on the growth and differentiation of NB4 cells NB4 cells were transfected with the pCDH-RA2 plasmid and the corresponding void vector, pCDH . Two distinct RARα2 expressing ( pRA2-NB4a and pRA2-NB4b ) and two ( pCDH-NB4a and pCDH-NB4b ) cell populations were isolated. A . Cells were treated with ATRA (1 μM) for 24 hours. Cell extracts were immuno-precipitated with an anti-RARα2 antibody [Ab25alpha2(A2)] coupled to Protein G sepharose beads. The immuno-precipitates were subjected to Western blot analysis with a different anti-RARα antibody [RP alpha (F)]. Equivalent amounts of protein extracts were used to immuno-precipitate RARα2, as indicated by the levels of actin present in the extracts before addition of the anti-RARα2 antibody (input). B . Cells were treated with vehicle (DMSO) or ATRA (1 μM) for the indicated amount of time. The number of viable cells determined after staining with trypan blue is indicated. Each point is the mean±S.D. of three replicate cultures. C . Cells were grown in the presence of vehicle (DMSO) or ATRA (1 μM) for 72 hours and subjected to FACS analysis for the determination of CD11b and CD11c. The column graphs indicate the MAF (mean-associated-fluorescence) values determined. D . Cells were grown as in (C) and treated with vehicle (DMSO) or ATRA (1 μM) for 48 hours. Cell extracts were subjected to Western blot analysis for the indicated proteins. Actin is used as a loading control. The calculated molecular weight (M.W.) of each protein is indicated on the left.
Figure Legend Snippet: Effects of RARα2 over-expression on the growth and differentiation of NB4 cells NB4 cells were transfected with the pCDH-RA2 plasmid and the corresponding void vector, pCDH . Two distinct RARα2 expressing ( pRA2-NB4a and pRA2-NB4b ) and two ( pCDH-NB4a and pCDH-NB4b ) cell populations were isolated. A . Cells were treated with ATRA (1 μM) for 24 hours. Cell extracts were immuno-precipitated with an anti-RARα2 antibody [Ab25alpha2(A2)] coupled to Protein G sepharose beads. The immuno-precipitates were subjected to Western blot analysis with a different anti-RARα antibody [RP alpha (F)]. Equivalent amounts of protein extracts were used to immuno-precipitate RARα2, as indicated by the levels of actin present in the extracts before addition of the anti-RARα2 antibody (input). B . Cells were treated with vehicle (DMSO) or ATRA (1 μM) for the indicated amount of time. The number of viable cells determined after staining with trypan blue is indicated. Each point is the mean±S.D. of three replicate cultures. C . Cells were grown in the presence of vehicle (DMSO) or ATRA (1 μM) for 72 hours and subjected to FACS analysis for the determination of CD11b and CD11c. The column graphs indicate the MAF (mean-associated-fluorescence) values determined. D . Cells were grown as in (C) and treated with vehicle (DMSO) or ATRA (1 μM) for 48 hours. Cell extracts were subjected to Western blot analysis for the indicated proteins. Actin is used as a loading control. The calculated molecular weight (M.W.) of each protein is indicated on the left.

Techniques Used: Over Expression, Transfection, Plasmid Preparation, Expressing, Isolation, Western Blot, Staining, FACS, Fluorescence, Molecular Weight

Expression, ATRA-dependent proteolytic degradation and transcriptional activity of PML-RAR, RARα2 and RARα1 A . NB4 cells were treated with vehicle (DMSO) or ATRA (0.1 μM) for 48 hours. Total RNA was extracted and subjected to RT-PCR analysis using Taqman assays for the indicated mRNAs. The results are expressed as the mean±SD of 3 replicates. B . Upper: NB4 cells were treated with vehicle (DMSO) or ATRA (0.1 μM) for 40 hours before addition of the proteasome inhibitor, MG132 (40 μM) for 8 hours. Total protein extracts were subjected to Western blot analysis with an anti-RARα antibody [RP alpha (F)]. Actin was used as a loading control. Lower: NB4 cells were treated as above with vehicle (DMSO), ATRA (0.1 μM), the proteasome inhibitor, MG132 (20 and 40 μM) or ATRA+MG132. Cell extracts were immuno-precipitated with an anti-RARα2 antibody [Ab25alpha2(A2)] coupled to protein G-sepharose beads (IP = immuno-precipitation) and the immuno-precipitates were subjected to Western blot analysis with the same anti-RARα antibody used in the Upper panel. Equivalent amounts of protein extracts were used to immuno-precipitate RARα2, as indicated by the levels of actin in the extracts before addition of the anti-RARα2 antibody (input). COS-7 = Total extracts of COS-7 cells transfected with a pcDNA3-RARα2 plasmid. The calculated molecular weight (M.W.) of the indicated proteins is shown on the left. C . COS-7 cells were transfected with pcDNA3-RARα2 , pcDNA3-RARα1 and pSG5-PML-RAR plasmids and the retinoid dependent Luciferase reporter, β2RARE-Luc . Sixteen hours following transfection, cells were treated with DMSO or the indicated concentrations of ATRA for an extra 24 hours. Cell extracts were used for the measurement of luciferase activity and the indicated proteins by Western blot analysis. Luciferase activity data are expressed as the mean±SD of two replicates. D . COS-7 cells were transfected as in ( C ). Sixteen hours following transfection, cells were treated with vehicle (DMSO) or ATRA (1 μM ) for 16 hours and vehicle or MG132 (40 μM) for an extra 8 hours. Cell extracts were used for the measurement of luciferase activity and the indicated proteins by Western blot analysis. Luciferase activity data are expressed as the mean±SD of two replicates.
Figure Legend Snippet: Expression, ATRA-dependent proteolytic degradation and transcriptional activity of PML-RAR, RARα2 and RARα1 A . NB4 cells were treated with vehicle (DMSO) or ATRA (0.1 μM) for 48 hours. Total RNA was extracted and subjected to RT-PCR analysis using Taqman assays for the indicated mRNAs. The results are expressed as the mean±SD of 3 replicates. B . Upper: NB4 cells were treated with vehicle (DMSO) or ATRA (0.1 μM) for 40 hours before addition of the proteasome inhibitor, MG132 (40 μM) for 8 hours. Total protein extracts were subjected to Western blot analysis with an anti-RARα antibody [RP alpha (F)]. Actin was used as a loading control. Lower: NB4 cells were treated as above with vehicle (DMSO), ATRA (0.1 μM), the proteasome inhibitor, MG132 (20 and 40 μM) or ATRA+MG132. Cell extracts were immuno-precipitated with an anti-RARα2 antibody [Ab25alpha2(A2)] coupled to protein G-sepharose beads (IP = immuno-precipitation) and the immuno-precipitates were subjected to Western blot analysis with the same anti-RARα antibody used in the Upper panel. Equivalent amounts of protein extracts were used to immuno-precipitate RARα2, as indicated by the levels of actin in the extracts before addition of the anti-RARα2 antibody (input). COS-7 = Total extracts of COS-7 cells transfected with a pcDNA3-RARα2 plasmid. The calculated molecular weight (M.W.) of the indicated proteins is shown on the left. C . COS-7 cells were transfected with pcDNA3-RARα2 , pcDNA3-RARα1 and pSG5-PML-RAR plasmids and the retinoid dependent Luciferase reporter, β2RARE-Luc . Sixteen hours following transfection, cells were treated with DMSO or the indicated concentrations of ATRA for an extra 24 hours. Cell extracts were used for the measurement of luciferase activity and the indicated proteins by Western blot analysis. Luciferase activity data are expressed as the mean±SD of two replicates. D . COS-7 cells were transfected as in ( C ). Sixteen hours following transfection, cells were treated with vehicle (DMSO) or ATRA (1 μM ) for 16 hours and vehicle or MG132 (40 μM) for an extra 8 hours. Cell extracts were used for the measurement of luciferase activity and the indicated proteins by Western blot analysis. Luciferase activity data are expressed as the mean±SD of two replicates.

Techniques Used: Expressing, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunoprecipitation, Transfection, Plasmid Preparation, Molecular Weight, Luciferase

10) Product Images from "Complexes between the LKB1 tumor suppressor, STRAD?/? and MO25?/? are upstream kinases in the AMP-activated protein kinase cascade"

Article Title: Complexes between the LKB1 tumor suppressor, STRAD?/? and MO25?/? are upstream kinases in the AMP-activated protein kinase cascade

Journal: Journal of Biology

doi: 10.1186/1475-4924-2-28

Endogenous AMPKK activity (that is, ability to activate AMPKα1 catalytic domain) can be immunoprecipitated from 293 cells using anti-LKB1 antibody, but activity can only be immunoprecipitated from HeLa cells if they stably express wild-type LKB1, but not a catalytically-inactive mutant. (a) LKB1 was immunoprecipitated from 0.5 mg cell extract derived from untransfected HEK-293T cells (lanes 1,2), untransfected HeLa cells (control; lanes 3,4), or HeLa cells stably expressing wild-type LKB1 (WT; lanes 5,6) or a kinase-dead LKB1 mutant (D194A; KD, lanes 7,8). Immunoprecipitation used anti-LKB1 (lanes 1, 3, 5, 7) or a pre-immune control immunoglobulin (IgG; lanes 2, 4, 6, 8). Samples of each immunoprecipitate were used to assay activation of GST-AMPKα1 catalytic domain, to analyze phosphorylation of GST-AMPKα1 catalytic domain on Thr172 (middle panel), and to determine by western blotting the recovery of LKB1 and its accessory subunits (bottom panels). In lanes 5 and 7 some immunoglobulin heavy chain (IgG-H) had eluted from the protein G-Sepharose despite the fact that it had been cross-linked: this explains why LKB1 may not appear to comigrate in lanes 1, 5 and 7. Also shown at left in the top panel is the basal activity obtained when the GST-AMPKα1-catalytic domain was incubated with MgATP on its own (no addition). (b) Whole cell lysates from the same cells were analyzed by SDS gel electrophoresis and blots probed using anti-LKB1, anti-STRADα, and anti-MO25α antibodies. They were also probed with anti-ERK1/2 antibodies as loading controls.
Figure Legend Snippet: Endogenous AMPKK activity (that is, ability to activate AMPKα1 catalytic domain) can be immunoprecipitated from 293 cells using anti-LKB1 antibody, but activity can only be immunoprecipitated from HeLa cells if they stably express wild-type LKB1, but not a catalytically-inactive mutant. (a) LKB1 was immunoprecipitated from 0.5 mg cell extract derived from untransfected HEK-293T cells (lanes 1,2), untransfected HeLa cells (control; lanes 3,4), or HeLa cells stably expressing wild-type LKB1 (WT; lanes 5,6) or a kinase-dead LKB1 mutant (D194A; KD, lanes 7,8). Immunoprecipitation used anti-LKB1 (lanes 1, 3, 5, 7) or a pre-immune control immunoglobulin (IgG; lanes 2, 4, 6, 8). Samples of each immunoprecipitate were used to assay activation of GST-AMPKα1 catalytic domain, to analyze phosphorylation of GST-AMPKα1 catalytic domain on Thr172 (middle panel), and to determine by western blotting the recovery of LKB1 and its accessory subunits (bottom panels). In lanes 5 and 7 some immunoglobulin heavy chain (IgG-H) had eluted from the protein G-Sepharose despite the fact that it had been cross-linked: this explains why LKB1 may not appear to comigrate in lanes 1, 5 and 7. Also shown at left in the top panel is the basal activity obtained when the GST-AMPKα1-catalytic domain was incubated with MgATP on its own (no addition). (b) Whole cell lysates from the same cells were analyzed by SDS gel electrophoresis and blots probed using anti-LKB1, anti-STRADα, and anti-MO25α antibodies. They were also probed with anti-ERK1/2 antibodies as loading controls.

Techniques Used: Activity Assay, Immunoprecipitation, Stable Transfection, Mutagenesis, Derivative Assay, Expressing, Activation Assay, Western Blot, Incubation, SDS-Gel, Electrophoresis

AMPKK activity (that is, the ability to activate AMPKα1 catalytic domain), and LKB1, STRADα and MO25α polypeptides, can be immunoprecipitated from rat liver AMPKK1 and AMPKK2 using anti-LKB1 antibody. (a) Depletion of AMPKK activity from supernatant. Sheep anti-human LKB1 or pre-immune control immunoglobulin (IgG) was prebound to Protein G-Sepharose beads and cross-linked with dimethylpimelimidate as described [ 47 ], except that a final wash of the beads with 100 mM glycine, pH 2.5, was performed. Bead-bound antibodies (40 μl) were incubated with the peak fraction of AMPKK1 (0.04 units), AMPKK2 (0.03 units) or recombinant GST-LKB1:STRADα:MO25α complex (0.06 units) for 120 minutes and the beads removed in a microcentrifuge (14,000 × g for 2 min). AMPKK activity was determined in the supernatants and is expressed as a percentage of the value obtained using the control IgG. (b) The pellets from the experiment in (a) were resuspended in the original volume and samples of the supernatants and pellets analysed by western blotting with anti-LKB1 antibody. The recombinant LKB1 migrates at a higher molecular mass because of the GST tag. (c) As in (a), except that the amounts of AMPKK1, AMPKK2 and recombinant GST-LKB1:STRADα:MO25α complex were increased to 0.44, 0.70 and 1.4 units, respectively, and the activities were determined in the resuspended pellets. In this experiment the amount of antibody was limiting, so only a fraction of the activity was precipitated. (d) The pellets from the experiment in (c) were resuspended and samples analyzed by western blotting with anti-LKB1, anti-STRADα and anti-MO25α antibodies.
Figure Legend Snippet: AMPKK activity (that is, the ability to activate AMPKα1 catalytic domain), and LKB1, STRADα and MO25α polypeptides, can be immunoprecipitated from rat liver AMPKK1 and AMPKK2 using anti-LKB1 antibody. (a) Depletion of AMPKK activity from supernatant. Sheep anti-human LKB1 or pre-immune control immunoglobulin (IgG) was prebound to Protein G-Sepharose beads and cross-linked with dimethylpimelimidate as described [ 47 ], except that a final wash of the beads with 100 mM glycine, pH 2.5, was performed. Bead-bound antibodies (40 μl) were incubated with the peak fraction of AMPKK1 (0.04 units), AMPKK2 (0.03 units) or recombinant GST-LKB1:STRADα:MO25α complex (0.06 units) for 120 minutes and the beads removed in a microcentrifuge (14,000 × g for 2 min). AMPKK activity was determined in the supernatants and is expressed as a percentage of the value obtained using the control IgG. (b) The pellets from the experiment in (a) were resuspended in the original volume and samples of the supernatants and pellets analysed by western blotting with anti-LKB1 antibody. The recombinant LKB1 migrates at a higher molecular mass because of the GST tag. (c) As in (a), except that the amounts of AMPKK1, AMPKK2 and recombinant GST-LKB1:STRADα:MO25α complex were increased to 0.44, 0.70 and 1.4 units, respectively, and the activities were determined in the resuspended pellets. In this experiment the amount of antibody was limiting, so only a fraction of the activity was precipitated. (d) The pellets from the experiment in (c) were resuspended and samples analyzed by western blotting with anti-LKB1, anti-STRADα and anti-MO25α antibodies.

Techniques Used: Activity Assay, Immunoprecipitation, Incubation, Recombinant, Western Blot

Recombinant LKB1:STRAD:MO25 complexes can efficiently activate the AMPKα1 catalytic domain via phosphorylation at Thr172. (a) The indicated combinations of GST-tagged wild-type LKB1 (WT, lanes 1–9), or kinase-dead (D194A; KD, lanes 10–13) LKB1 mutant, or GST-alone (lane 14), FLAG-tagged STRADα or STRADβ, and Myc-tagged MO25α or MO25β were coexpressed in HEK-293T cells, purified on glutathione-Sepharose and tested for their ability to activate GST-AMPKα1 catalytic domain (top panel). The results are expressed as the increase in the units of AMPK activity generated per mg full-length GST-AMPKα1 catalytic domain. Samples from each incubation were also analyzed by western blotting and probed using the indicated antibodies (from top to bottom): anti-pT172; anti-AMPKα1 catalytic domain (GST-AMPKα1); anti-GST to detect GST-LKB1; anti-FLAG to detect STRADα and STRADβ, and anti-Myc to detect MO25α and MO25β. All proteins migrated with the expected mobility, taking into account the epitope tags. The bottom three blots were conducted on blank reactions lacking GST-AMPKα1 catalytic domain, as the latter appeared to cause some interference with detection. (b) Recombinant GST-LKB1:STRADα:MO25α complex was used to phosphorylate wild-type GST-AMPKα1 catalytic domain (GST-α1-WT) or a T172A mutant (GST-α1-T172A) using [γ- 32 P]ATP as described in Materials and methods. The reaction was analyzed by SDS gel electrophoresis and autoradiography. Arrows show the migration of GST-LKB1 (which autophosphorylates) and GST-AMPKα1 catalytic domain.
Figure Legend Snippet: Recombinant LKB1:STRAD:MO25 complexes can efficiently activate the AMPKα1 catalytic domain via phosphorylation at Thr172. (a) The indicated combinations of GST-tagged wild-type LKB1 (WT, lanes 1–9), or kinase-dead (D194A; KD, lanes 10–13) LKB1 mutant, or GST-alone (lane 14), FLAG-tagged STRADα or STRADβ, and Myc-tagged MO25α or MO25β were coexpressed in HEK-293T cells, purified on glutathione-Sepharose and tested for their ability to activate GST-AMPKα1 catalytic domain (top panel). The results are expressed as the increase in the units of AMPK activity generated per mg full-length GST-AMPKα1 catalytic domain. Samples from each incubation were also analyzed by western blotting and probed using the indicated antibodies (from top to bottom): anti-pT172; anti-AMPKα1 catalytic domain (GST-AMPKα1); anti-GST to detect GST-LKB1; anti-FLAG to detect STRADα and STRADβ, and anti-Myc to detect MO25α and MO25β. All proteins migrated with the expected mobility, taking into account the epitope tags. The bottom three blots were conducted on blank reactions lacking GST-AMPKα1 catalytic domain, as the latter appeared to cause some interference with detection. (b) Recombinant GST-LKB1:STRADα:MO25α complex was used to phosphorylate wild-type GST-AMPKα1 catalytic domain (GST-α1-WT) or a T172A mutant (GST-α1-T172A) using [γ- 32 P]ATP as described in Materials and methods. The reaction was analyzed by SDS gel electrophoresis and autoradiography. Arrows show the migration of GST-LKB1 (which autophosphorylates) and GST-AMPKα1 catalytic domain.

Techniques Used: Recombinant, Mutagenesis, Purification, Activity Assay, Generated, Incubation, Western Blot, SDS-Gel, Electrophoresis, Autoradiography, Migration

Two AMPKKs can be resolved from rat liver extracts and both contain LKB1, STRADα and MO25α. (a) Separation of two activities that activate the GST-AMPKα1 catalytic domain by Q-Sepharose chromatography. The graph shows AMPKK activity in 4.5 ml fractions (red circles and red line), absorbance at 280 nm (continuous black line) and conductivity in the eluate (dashed black line) plotted against fraction number. (b) Probing of blots of column fractions after SDS gel electrophoresis (1 μl per lane) using anti-LKB1, anti-STRADα or anti-MO25α antibodies. In the three bottom panels, fractions 26–30 were concentrated from 4.5 ml to 250 μl using Amicon Ultra-4 30,000 MWCO centrifugal concentrators, and reanalyzed by western blotting using 2 μl per lane. (c) The effect of protein phosphatase treatment on the mobility of LKB1. The peak fractions of AMPKK1 (0.2 units) or AMPKK2 (0.8 units) were incubated in a final volume of 20 μl with or without 5 mM MgCl2 and 200 μM ATP for 15 min at 30°C. Protein phosphatases (PP1γ, 8 mU; or PP2A 1 , 1 mU) or buffer were added and incubation continued for a further 15 min before stopping the reactions in SDS sample buffer and analyzing by SDS gel electrophoresis and western blotting using anti-LKB1 antibody.
Figure Legend Snippet: Two AMPKKs can be resolved from rat liver extracts and both contain LKB1, STRADα and MO25α. (a) Separation of two activities that activate the GST-AMPKα1 catalytic domain by Q-Sepharose chromatography. The graph shows AMPKK activity in 4.5 ml fractions (red circles and red line), absorbance at 280 nm (continuous black line) and conductivity in the eluate (dashed black line) plotted against fraction number. (b) Probing of blots of column fractions after SDS gel electrophoresis (1 μl per lane) using anti-LKB1, anti-STRADα or anti-MO25α antibodies. In the three bottom panels, fractions 26–30 were concentrated from 4.5 ml to 250 μl using Amicon Ultra-4 30,000 MWCO centrifugal concentrators, and reanalyzed by western blotting using 2 μl per lane. (c) The effect of protein phosphatase treatment on the mobility of LKB1. The peak fractions of AMPKK1 (0.2 units) or AMPKK2 (0.8 units) were incubated in a final volume of 20 μl with or without 5 mM MgCl2 and 200 μM ATP for 15 min at 30°C. Protein phosphatases (PP1γ, 8 mU; or PP2A 1 , 1 mU) or buffer were added and incubation continued for a further 15 min before stopping the reactions in SDS sample buffer and analyzing by SDS gel electrophoresis and western blotting using anti-LKB1 antibody.

Techniques Used: Chromatography, Activity Assay, SDS-Gel, Electrophoresis, Western Blot, Incubation

11) Product Images from "The V Protein of Tioman Virus Is Incapable of Blocking Type I Interferon Signaling in Human Cells"

Article Title: The V Protein of Tioman Virus Is Incapable of Blocking Type I Interferon Signaling in Human Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0053881

TioV-V binds MDA5, LGP2, and STAT3. ( A–C ) HEK-293T cells were co-transfected with expression vectors encoding GST alone or fused to MuV-V ( A–C ), TioV-V ( A–F ), TioV-W ( D–F ) or TioV-VCT ( D–F ) (500 ng/well), and pCI-neo-3xFLAG expression vectors (300 ng/well) encoding for 3xFLAG-tagged human MDA5 ( A and D ), LGP2 ( B and E ), STAT3 ( C and F ). Total cell lysates from transfected cells were prepared at 48 h post-transfection (cell lysate; middle and lower panels), and protein complexes were assayed by pull-down using glutathione-sepharose beads (GST pull-down; upper panel). 3xFLAG- and GST-tagged proteins were detected by immunoblotting.
Figure Legend Snippet: TioV-V binds MDA5, LGP2, and STAT3. ( A–C ) HEK-293T cells were co-transfected with expression vectors encoding GST alone or fused to MuV-V ( A–C ), TioV-V ( A–F ), TioV-W ( D–F ) or TioV-VCT ( D–F ) (500 ng/well), and pCI-neo-3xFLAG expression vectors (300 ng/well) encoding for 3xFLAG-tagged human MDA5 ( A and D ), LGP2 ( B and E ), STAT3 ( C and F ). Total cell lysates from transfected cells were prepared at 48 h post-transfection (cell lysate; middle and lower panels), and protein complexes were assayed by pull-down using glutathione-sepharose beads (GST pull-down; upper panel). 3xFLAG- and GST-tagged proteins were detected by immunoblotting.

Techniques Used: Transfection, Expressing

TioV-V binds DDB1. (A–B) HEK-293T cells were co-transfected with expression vectors encoding GST alone or fused to MuV-V ( A ), TioV-V ( A–B ), TioV-W ( B ) or TioV-VCT ( B ) (500 ng/well), and pCI-neo-3xFLAG expression vectors (300 ng/well) encoding for 3xFLAG-tagged human DDB1 ( A–B ). Total cell lysates from transfected cells were prepared at 48 h post-transfection (cell lysate; middle and lower panels), and protein complexes were assayed by pull-down using glutathione-sepharose beads (GST pull-down; upper panel). 3xFLAG- and GST-tagged proteins were detected by immunoblotting.
Figure Legend Snippet: TioV-V binds DDB1. (A–B) HEK-293T cells were co-transfected with expression vectors encoding GST alone or fused to MuV-V ( A ), TioV-V ( A–B ), TioV-W ( B ) or TioV-VCT ( B ) (500 ng/well), and pCI-neo-3xFLAG expression vectors (300 ng/well) encoding for 3xFLAG-tagged human DDB1 ( A–B ). Total cell lysates from transfected cells were prepared at 48 h post-transfection (cell lysate; middle and lower panels), and protein complexes were assayed by pull-down using glutathione-sepharose beads (GST pull-down; upper panel). 3xFLAG- and GST-tagged proteins were detected by immunoblotting.

Techniques Used: Transfection, Expressing

TioV-V fails to interact with human STAT2 and does not induce STAT1 degradation. ( A–B ) HEK-293T cells were co-transfected with expression vectors encoding GST alone or fused to MuV-V, TioV-V ( A–B ) or NiV-V ( B ) (500 ng/well), and pCI-neo-3xFLAG expression vectors (300 ng/well) encoding for 3xFLAG-tagged human STAT2 ( A ) or STAT1 ( B ). Total cell lysates from transfected cells were prepared at 48 h post-transfection (cell lysate; middle and lower panels), and protein complexes were assayed by pull-down using glutathione-sepharose beads (GST pull-down; upper panel). 3xFLAG- and GST-tagged proteins were detected by immunoblotting. ( C ) HEK-293T cells were co-transfected with expression vectors encoding GST alone or fused to TioV-V, MuV-V or CHIKV-nsP4 (500 ng/well), and pCI-neo-3xFLAG expression vectors encoding for 3xFLAG-tagged human STAT1 and STAT2 (150 ng/well of each vector). At 24 h post-transfection, cells were left untreated or stimulated with recombinant IFN-β at 200 IU/ml. Total cell lysates from transfected cells were prepared at 48 h post-transfection (cell lysate; middle and lower panels), and protein complexes were assayed by pull-down using glutathione-sepharose beads (GST pull-down; upper panels). 3xFLAG- and GST-tagged proteins were detected by immunoblotting. Upper and lower panels on top of figure C correspond to short and longer exposures of the same blot, respectively. ( D ) HEK-293T cells were transfected with pCI-neo-3xFLAG expression vector (1 µg/well) either empty or encoding for 3xFLAG-tagged TioV-V, MuV-V, TioV-P or TioV-W. Total cell lysates were prepared at 48 h post-transfection and endogenous STAT1 expression levels were determined by western-blot analysis. Actin expression was determined and used as a protein extraction and loading control. ( E ) HEK-293T cells were transfected with pCI-neo-3xFLAG expression vector (1 µg/well) either empty or encoding for 3xFLAG-tagged TioV-V, MuV-V or CHIKV-nsP4. Total cell lysates were prepared at 48 h post-transfection, and 3xFLAG-tagged viral proteins were purified using anti-FLAG antibodies conjugated to sepharose beads. Co-immunopurification of endogenous STAT2 with 3xFLAG-tagged viral proteins was determined by western-blot analysis (top and middle panel, respectively). Actin expression was determined prior to the immunoprecipitation on total cell lysates and used as a protein extraction control (lower panel).
Figure Legend Snippet: TioV-V fails to interact with human STAT2 and does not induce STAT1 degradation. ( A–B ) HEK-293T cells were co-transfected with expression vectors encoding GST alone or fused to MuV-V, TioV-V ( A–B ) or NiV-V ( B ) (500 ng/well), and pCI-neo-3xFLAG expression vectors (300 ng/well) encoding for 3xFLAG-tagged human STAT2 ( A ) or STAT1 ( B ). Total cell lysates from transfected cells were prepared at 48 h post-transfection (cell lysate; middle and lower panels), and protein complexes were assayed by pull-down using glutathione-sepharose beads (GST pull-down; upper panel). 3xFLAG- and GST-tagged proteins were detected by immunoblotting. ( C ) HEK-293T cells were co-transfected with expression vectors encoding GST alone or fused to TioV-V, MuV-V or CHIKV-nsP4 (500 ng/well), and pCI-neo-3xFLAG expression vectors encoding for 3xFLAG-tagged human STAT1 and STAT2 (150 ng/well of each vector). At 24 h post-transfection, cells were left untreated or stimulated with recombinant IFN-β at 200 IU/ml. Total cell lysates from transfected cells were prepared at 48 h post-transfection (cell lysate; middle and lower panels), and protein complexes were assayed by pull-down using glutathione-sepharose beads (GST pull-down; upper panels). 3xFLAG- and GST-tagged proteins were detected by immunoblotting. Upper and lower panels on top of figure C correspond to short and longer exposures of the same blot, respectively. ( D ) HEK-293T cells were transfected with pCI-neo-3xFLAG expression vector (1 µg/well) either empty or encoding for 3xFLAG-tagged TioV-V, MuV-V, TioV-P or TioV-W. Total cell lysates were prepared at 48 h post-transfection and endogenous STAT1 expression levels were determined by western-blot analysis. Actin expression was determined and used as a protein extraction and loading control. ( E ) HEK-293T cells were transfected with pCI-neo-3xFLAG expression vector (1 µg/well) either empty or encoding for 3xFLAG-tagged TioV-V, MuV-V or CHIKV-nsP4. Total cell lysates were prepared at 48 h post-transfection, and 3xFLAG-tagged viral proteins were purified using anti-FLAG antibodies conjugated to sepharose beads. Co-immunopurification of endogenous STAT2 with 3xFLAG-tagged viral proteins was determined by western-blot analysis (top and middle panel, respectively). Actin expression was determined prior to the immunoprecipitation on total cell lysates and used as a protein extraction control (lower panel).

Techniques Used: Transfection, Expressing, Plasmid Preparation, Recombinant, Western Blot, Protein Extraction, Purification, Immu-Puri, Immunoprecipitation

TioV-V fails to interact with Pr STAT2. HEK-293T cells were co-transfected with expression vectors encoding GST alone or fused to TioV-V or MuV-V (500 ng/well), and pCI-neo-3xFLAG expression vectors (300 ng/well) encoding for 3xFLAG-tagged STAT2 from human (hSTAT2) or Pteropus rodricensis ( Pr STAT2). Total cell lysates from transfected cells were prepared 48 h post-transfection (cell lysate; middle and lower panels), and protein complexes were assayed by pull-down using glutathione-sepharose beads (GST pull-down; upper panel). 3xFLAG- and GST-tagged proteins were detected by immunoblotting.
Figure Legend Snippet: TioV-V fails to interact with Pr STAT2. HEK-293T cells were co-transfected with expression vectors encoding GST alone or fused to TioV-V or MuV-V (500 ng/well), and pCI-neo-3xFLAG expression vectors (300 ng/well) encoding for 3xFLAG-tagged STAT2 from human (hSTAT2) or Pteropus rodricensis ( Pr STAT2). Total cell lysates from transfected cells were prepared 48 h post-transfection (cell lysate; middle and lower panels), and protein complexes were assayed by pull-down using glutathione-sepharose beads (GST pull-down; upper panel). 3xFLAG- and GST-tagged proteins were detected by immunoblotting.

Techniques Used: Transfection, Expressing

12) Product Images from "Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5 *"

Article Title: Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5 *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M807055200

The cytosolic C terminus of NHE5 interacts directly with SCAMP2. A , GST or GST fusion proteins containing either the cytoplasmic N terminus (amino acids 1-154), C terminus (amino acids 284-329), or the cytosolic loop ( CL , amino acids 201-215) between the second and third trans-membrane domains of SCAMP2 were immobilized on reduced glutathione-Sepharose beads. The beads were then incubated with 35 S-labeled in vitro transcribed/translated NHE5 C terminus (amino acids 492-896). After washing the beads, bound 35 S-labeled NHE5 C terminus was eluted, resolved by SDS-PAGE, and detected by phosphorimaging. A small amount of the NHE5 C terminus input (3%), not subjected to pulldown assay, was also included as a control. B and C , two additional GST pulldown experiments were performed using GST fused to fragments of the SCAMP2 N terminus (amino acids 1-154, 1-88, 45-88, 45-154, 75-117, 75-134, and 75-154 in B , or amino acids 1-154, 1-44, and 134-154 in C ) to determine the minimum NHE5-binding sites within the SCAMP2 N-terminal tail. Each pulldown experiment was performed three times; representative results are shown. D , a schematic representation showing the membrane topology of SCAMP2. NHE5-binding sites are highlighted with black rectangles , and the N-terminal NPF repeats are labeled with black circles . Numbers indicate amino acid residues.
Figure Legend Snippet: The cytosolic C terminus of NHE5 interacts directly with SCAMP2. A , GST or GST fusion proteins containing either the cytoplasmic N terminus (amino acids 1-154), C terminus (amino acids 284-329), or the cytosolic loop ( CL , amino acids 201-215) between the second and third trans-membrane domains of SCAMP2 were immobilized on reduced glutathione-Sepharose beads. The beads were then incubated with 35 S-labeled in vitro transcribed/translated NHE5 C terminus (amino acids 492-896). After washing the beads, bound 35 S-labeled NHE5 C terminus was eluted, resolved by SDS-PAGE, and detected by phosphorimaging. A small amount of the NHE5 C terminus input (3%), not subjected to pulldown assay, was also included as a control. B and C , two additional GST pulldown experiments were performed using GST fused to fragments of the SCAMP2 N terminus (amino acids 1-154, 1-88, 45-88, 45-154, 75-117, 75-134, and 75-154 in B , or amino acids 1-154, 1-44, and 134-154 in C ) to determine the minimum NHE5-binding sites within the SCAMP2 N-terminal tail. Each pulldown experiment was performed three times; representative results are shown. D , a schematic representation showing the membrane topology of SCAMP2. NHE5-binding sites are highlighted with black rectangles , and the N-terminal NPF repeats are labeled with black circles . Numbers indicate amino acid residues.

Techniques Used: Incubation, Labeling, In Vitro, SDS Page, Binding Assay

Arf6 and Rab11 up-regulate NHE5 cell-surface targeting and activity. A and B , AP-1/NHE5 HA cells transfected with empty vector (pcDNA3), wild-type Arf6 HA ( Arf6 WT ), Arf6T27N HA ( Arf6 T27N ), Arf6T27N HA plus Rab11S25N Myc ( Arf6 T27N / Rab11 S25N ), wild-type Rab11 Myc ( Rab11 WT ) or Rab11S25N Myc ( Rab11 S25N ) were subjected to surface labeling using a protein reactive biotinylation reagent. Labeled proteins were isolated from cell lysates by incubation with avidin-coupled agarose beads, and biotinylated NHE5 HA was analyzed by Western blot ( Surface NHE5 HA ). A five percent volume of the lysate, not subjected to avidin-coupled beads, was analyzed by Western blot as a loading control ( Total NHE5 HA , Rab11 Myc , and Arf6 HA ). A , representative Western blots. B , surface NHE5 HA from the different transfection conditions was measured by densitometry and is expressed relative to pcDNA3-transfected control ± S.D. Data are averaged from five independent experiments, asterisks represent statistical significance, p
Figure Legend Snippet: Arf6 and Rab11 up-regulate NHE5 cell-surface targeting and activity. A and B , AP-1/NHE5 HA cells transfected with empty vector (pcDNA3), wild-type Arf6 HA ( Arf6 WT ), Arf6T27N HA ( Arf6 T27N ), Arf6T27N HA plus Rab11S25N Myc ( Arf6 T27N / Rab11 S25N ), wild-type Rab11 Myc ( Rab11 WT ) or Rab11S25N Myc ( Rab11 S25N ) were subjected to surface labeling using a protein reactive biotinylation reagent. Labeled proteins were isolated from cell lysates by incubation with avidin-coupled agarose beads, and biotinylated NHE5 HA was analyzed by Western blot ( Surface NHE5 HA ). A five percent volume of the lysate, not subjected to avidin-coupled beads, was analyzed by Western blot as a loading control ( Total NHE5 HA , Rab11 Myc , and Arf6 HA ). A , representative Western blots. B , surface NHE5 HA from the different transfection conditions was measured by densitometry and is expressed relative to pcDNA3-transfected control ± S.D. Data are averaged from five independent experiments, asterisks represent statistical significance, p

Techniques Used: Activity Assay, Transfection, Plasmid Preparation, Labeling, Isolation, Incubation, Avidin-Biotin Assay, Western Blot

SCAMP2 controls NHE5 cell-surface abundance. A and B , AP-1/NHE5 HA cells were transiently transfected with Myc-tagged SCAMP2 (SCAMP2 Myc , A ) or Myc-tagged SCAMP5 (SCAMP5 Myc , B ), or with empty pcDNA3 vector control. Transfected cells were incubated with a biotinylation reagent followed by chase incubation in the culture media for 0, 15, or 30 min ( Chase ) to permit endocytosis of labeled proteins. Following the chase period, surface biotin was removed by incubation with a cleavage reagent allowing visualization of internalized protein or left uncleaved (Cleavage: + or -). Cells were then lysed and biotinylated proteins were purified by incubation with avidin-conjugated agarose beads, resolved by SDS-PAGE, and surface-labeled and -internalized NHE5 HA was detected by Western blotting using an anti-HA monoclonal antibody ( Surface NHE5 HA ). A small amount of total lysate (5%) was analyzed as a loading control and probed for SCAMP2 Myc or SCAMP5 Myc and NHE5 HA ( Total NHE5 HA , SCAMP2 Myc , or SCAMP5 Myc ). The Western blots shown are representative of three independent experiments. C , the percentage of labeled NHE5 HA internalized after 15 or 30 min of chase was calculated by comparing the signal in the cleaved samples (Cleavage: +) to the corresponding uncleaved samples (Cleavage: -). The amount of NHE5 HA internalized at each time point in SCAMP2 Myc - or SCAMP5 Myc -transfected cells is expressed relative to control cells (pcDNA3) and are averaged from three independent experiments ± S.D. D , densitometric analysis of the biotinylated samples without chase (time 0 min), representing total surface-labeled protein, was used to measure the relative surface abundance of NHE5 HA . Total surface NHE5 HA in SCAMP2 Myc - or SCAMP5 Myc -transfected cells was compared directly to pcDNA3-transfected control cells from the same experiment and is expressed as a percentage relative to pcDNA3 transfected control. Values represent the averaged result from three independent experiments ± S.D. ** and NS, p
Figure Legend Snippet: SCAMP2 controls NHE5 cell-surface abundance. A and B , AP-1/NHE5 HA cells were transiently transfected with Myc-tagged SCAMP2 (SCAMP2 Myc , A ) or Myc-tagged SCAMP5 (SCAMP5 Myc , B ), or with empty pcDNA3 vector control. Transfected cells were incubated with a biotinylation reagent followed by chase incubation in the culture media for 0, 15, or 30 min ( Chase ) to permit endocytosis of labeled proteins. Following the chase period, surface biotin was removed by incubation with a cleavage reagent allowing visualization of internalized protein or left uncleaved (Cleavage: + or -). Cells were then lysed and biotinylated proteins were purified by incubation with avidin-conjugated agarose beads, resolved by SDS-PAGE, and surface-labeled and -internalized NHE5 HA was detected by Western blotting using an anti-HA monoclonal antibody ( Surface NHE5 HA ). A small amount of total lysate (5%) was analyzed as a loading control and probed for SCAMP2 Myc or SCAMP5 Myc and NHE5 HA ( Total NHE5 HA , SCAMP2 Myc , or SCAMP5 Myc ). The Western blots shown are representative of three independent experiments. C , the percentage of labeled NHE5 HA internalized after 15 or 30 min of chase was calculated by comparing the signal in the cleaved samples (Cleavage: +) to the corresponding uncleaved samples (Cleavage: -). The amount of NHE5 HA internalized at each time point in SCAMP2 Myc - or SCAMP5 Myc -transfected cells is expressed relative to control cells (pcDNA3) and are averaged from three independent experiments ± S.D. D , densitometric analysis of the biotinylated samples without chase (time 0 min), representing total surface-labeled protein, was used to measure the relative surface abundance of NHE5 HA . Total surface NHE5 HA in SCAMP2 Myc - or SCAMP5 Myc -transfected cells was compared directly to pcDNA3-transfected control cells from the same experiment and is expressed as a percentage relative to pcDNA3 transfected control. Values represent the averaged result from three independent experiments ± S.D. ** and NS, p

Techniques Used: Transfection, Plasmid Preparation, Incubation, Labeling, Purification, Avidin-Biotin Assay, SDS Page, Western Blot

NHE5 interacts with SCAMPs. A , membrane fractions from control PC12 cells or PC12 cells stably expressing NHE5 1D4 (PC12/NHE5 1D4 ) were immunoprecipitated with 1D4 antibody conjugated to Sepharose beads. Bound endogenous SCAMP1, SCAMP2, and SCAMP5 found in the immunoprecipitate fraction ( IP ) were detected by Western blot using SCAMP-specific antibodies. Five percent of the membrane lysate ( Lys. ) was resolved as a positive control. B , 2.5, 5, and 10 μg of protein from rat brain lysate was probed by Western blot to assess the endogenous expression of NHE5 and SCAMP2 protein in brain tissue. C , NHE5 was immunoprecipitated from rat brain lysate using an anti-NHE5 antibody ( IP ) or pre-immune serum control ( Con. ), and bound endogenous SCAMP2 was detected by Western blot. One percent of the rat brain lysate ( Lys. ) was probed as a positive control. Western blots shown in A-C are representative of three independent experiments in each case.
Figure Legend Snippet: NHE5 interacts with SCAMPs. A , membrane fractions from control PC12 cells or PC12 cells stably expressing NHE5 1D4 (PC12/NHE5 1D4 ) were immunoprecipitated with 1D4 antibody conjugated to Sepharose beads. Bound endogenous SCAMP1, SCAMP2, and SCAMP5 found in the immunoprecipitate fraction ( IP ) were detected by Western blot using SCAMP-specific antibodies. Five percent of the membrane lysate ( Lys. ) was resolved as a positive control. B , 2.5, 5, and 10 μg of protein from rat brain lysate was probed by Western blot to assess the endogenous expression of NHE5 and SCAMP2 protein in brain tissue. C , NHE5 was immunoprecipitated from rat brain lysate using an anti-NHE5 antibody ( IP ) or pre-immune serum control ( Con. ), and bound endogenous SCAMP2 was detected by Western blot. One percent of the rat brain lysate ( Lys. ) was probed as a positive control. Western blots shown in A-C are representative of three independent experiments in each case.

Techniques Used: Stable Transfection, Expressing, Immunoprecipitation, Western Blot, Positive Control

13) Product Images from "The third helix of the homeodomain of paired class homeodomain proteins acts as a recognition helix both for DNA and protein interactions"

Article Title: The third helix of the homeodomain of paired class homeodomain proteins acts as a recognition helix both for DNA and protein interactions

Journal: Nucleic Acids Research

doi: 10.1093/nar/gki562

The acidic residues E101, E112, E120, D123 and E128 in the paired domain are important for the interaction with the homeodomain of Pax6. GST pull-down assays with Pax6 HD fused to GST and immobilized on glutathione–agarose beads and Pax6ΔHD protein produced by in vitro transcription and translation in the presence of [ 35 S]methionine. ( A ) The single mutants of Pax6ΔHD, E112A, E120A and E128A bind to GST-HD with the same affinity as the wild type. For the double mutants the binding to the homeodomain was reduced by 40–60%. The triple mutant displays only 15% residual binding while the quadruple mutants showed 11 and 5% binding, respectively. The GST pull-downs were performed as described in the legend to Figure 1 . ( B ) Quantitative representation of the interaction data determined as described in the legend to Figure 3 . The data shown represent the mean of three independent experiments.
Figure Legend Snippet: The acidic residues E101, E112, E120, D123 and E128 in the paired domain are important for the interaction with the homeodomain of Pax6. GST pull-down assays with Pax6 HD fused to GST and immobilized on glutathione–agarose beads and Pax6ΔHD protein produced by in vitro transcription and translation in the presence of [ 35 S]methionine. ( A ) The single mutants of Pax6ΔHD, E112A, E120A and E128A bind to GST-HD with the same affinity as the wild type. For the double mutants the binding to the homeodomain was reduced by 40–60%. The triple mutant displays only 15% residual binding while the quadruple mutants showed 11 and 5% binding, respectively. The GST pull-downs were performed as described in the legend to Figure 1 . ( B ) Quantitative representation of the interaction data determined as described in the legend to Figure 3 . The data shown represent the mean of three independent experiments.

Techniques Used: Produced, In Vitro, Binding Assay, Mutagenesis

Mutation of basic amino acids in helix 3 of the Chx10 homeodomain leads to reduced interaction with and superactivation by Pax6ΔHD. ( A ) GST pull-down assays with Chx10 HD wild type and mutants fused to GST and immobilized on glutathione–agarose beads and Pax6ΔHD protein produced by in vitro transcription and translation in the presence of [ 35 S]methionine. ( B ) Quantitative representation of the interaction data determined as described in the legend to Figure 3 . ( C ) Effects of mutations in the recognition helix of the HD of Chx10 on superactivation of Pax6ΔHD-mediated transactivation from paired domain-binding sites. HeLa cells were co-transfected with 0.5 μg Pax6ΔHD, 0.5 μg pP6CON-LUC and 5 ng pCMV-βgal together with either 0.25 μg pcDNA3-HA vector, HAChx10 or HA-Chx10 mutants. HA-Chx10 co-transfected with the empty Pax6ΔHD control vector shows that Chx10 alone does not activate the P6CON LUC reporter. The data in (B) and (C) represent the mean of three independent experiments. ( D ) Western blot showing similar expression levels of wild type and all helix 3 mutants of Chx10 following transfection of HeLa cells. EGFP served as transfection control.
Figure Legend Snippet: Mutation of basic amino acids in helix 3 of the Chx10 homeodomain leads to reduced interaction with and superactivation by Pax6ΔHD. ( A ) GST pull-down assays with Chx10 HD wild type and mutants fused to GST and immobilized on glutathione–agarose beads and Pax6ΔHD protein produced by in vitro transcription and translation in the presence of [ 35 S]methionine. ( B ) Quantitative representation of the interaction data determined as described in the legend to Figure 3 . ( C ) Effects of mutations in the recognition helix of the HD of Chx10 on superactivation of Pax6ΔHD-mediated transactivation from paired domain-binding sites. HeLa cells were co-transfected with 0.5 μg Pax6ΔHD, 0.5 μg pP6CON-LUC and 5 ng pCMV-βgal together with either 0.25 μg pcDNA3-HA vector, HAChx10 or HA-Chx10 mutants. HA-Chx10 co-transfected with the empty Pax6ΔHD control vector shows that Chx10 alone does not activate the P6CON LUC reporter. The data in (B) and (C) represent the mean of three independent experiments. ( D ) Western blot showing similar expression levels of wild type and all helix 3 mutants of Chx10 following transfection of HeLa cells. EGFP served as transfection control.

Techniques Used: Mutagenesis, Produced, In Vitro, Binding Assay, Transfection, Plasmid Preparation, Western Blot, Expressing

Arginines (R44, R53 and R57) in the recognition helix and N-terminal arm (R3 and R5) of the homeodomain of Pax6 are important for the interaction with the paired domain. GST pull-down assays with Pax6 HD, and HD mutations fused to GST and immobilized on glutathione–agarose beads and in vitro translated Pax6ΔHD. ( A ) The GST pull-downs were performed as described in the legend to Figure 1 . The panel shows two different experiments using two different GST-HD fusions. The GST-HD protein (upper panel) contains two amino acids N-terminal to the HD while the GST-18L-HD (lower panel) contains 18 amino acids of the linker region N-terminal to the HD to study the effect of mutating also at −3 relative to the start of the HD. ( B ) Quantitative representation of the interaction data. A Fuji Bio-imaging analyzer (BAS5000) equipped with Image Gauge version 4.0 software was used to quantitate 35 S-labeled proteins in the SDS–polyacrylamide gels. The amount 35 S-labeled Pax6ΔHD pulled down by wild-type GST-HD was set to 100%. The data shown represent the mean of three independent experiments.
Figure Legend Snippet: Arginines (R44, R53 and R57) in the recognition helix and N-terminal arm (R3 and R5) of the homeodomain of Pax6 are important for the interaction with the paired domain. GST pull-down assays with Pax6 HD, and HD mutations fused to GST and immobilized on glutathione–agarose beads and in vitro translated Pax6ΔHD. ( A ) The GST pull-downs were performed as described in the legend to Figure 1 . The panel shows two different experiments using two different GST-HD fusions. The GST-HD protein (upper panel) contains two amino acids N-terminal to the HD while the GST-18L-HD (lower panel) contains 18 amino acids of the linker region N-terminal to the HD to study the effect of mutating also at −3 relative to the start of the HD. ( B ) Quantitative representation of the interaction data. A Fuji Bio-imaging analyzer (BAS5000) equipped with Image Gauge version 4.0 software was used to quantitate 35 S-labeled proteins in the SDS–polyacrylamide gels. The amount 35 S-labeled Pax6ΔHD pulled down by wild-type GST-HD was set to 100%. The data shown represent the mean of three independent experiments.

Techniques Used: In Vitro, Imaging, Software, Labeling

The recognition helix of the homeodomain of Pax6 is important for interaction with both the PD and the HD. ( A ) Pax6 constructs used for in vitro translation and GST pull-downs. ( B ) GST pull-down assays with Pax6 HD and PD fused to GST and immobilized on glutathione–agarose beads and Pax6ΔPDΔHD, Pax6ΔPDΔh2–3 or Pax6ΔPDΔh3 produced by in vitro transcription and translation in the presence of [ 35 S]methionine. An aliquot of 10 μl of the in vitro translation reactions was preincubated with GST immobilized on glutathione–agarose beads before incubation with the GST fusion proteins. The GST beads, GST-Pax6 HD beads and GST-Pax6 PD beads were washed several times before they were boiled in SDS loading buffer and run on a 10% SDS–polyacrylamide gel. An aliquot of 2 μl of the in vitro translated proteins was run on the same gel to visualize the signal from 20% of the input. ( C ) Point mutations in helix 3 of the homeodomain strongly reduce the ability of Pax6 HD to interact with the PD and the wild-type HD. The N51Q, R53A and R58A, but not S50A, mutants impede the HD–PD and HD–HD interactions. GST pull-down assays were performed with recombinant GST fusions of wild type or mutants of Pax6ΔHD against in vitro translated, [ 35 S]methionine-labeled Pax6, Pax6ΔHD or Pax6ΔPD. ( D ) The interactions between full-length Pax6 and the RED subdomain and between full-length Pax6 and the HD are independent of DNA. GST pull-down assays were done with Pax6 HD and RED fused to GST as in (C). Where indicated, the pull-down experiments were performed in the presence of 500 U benzonase to degrade both DNA and RNA. The results shown are representative of three independent experiments. ( E ) The PD–HD and HD–HD interactions of Pax6 are also observed in the yeast-based SOS recruitment interaction system. The temperature sensitive yeast strain S.cerevisiae cdc25-2 MATa was co-transformed either with pSOS-zfPax6-HDwt and empty pMYR or pMYR-LaminC as negative controls, pMYR-SOS binding protein as a positive control, pMYR-zfPax6-HDwt, or with pMYR-zfPax6-PDwt. Three independent colonies generated from each co-transformation were replica plated onto galactose plates and grown in parallel at 25 and 37°C for 6 days. The results shown are representative of three independent experiments.
Figure Legend Snippet: The recognition helix of the homeodomain of Pax6 is important for interaction with both the PD and the HD. ( A ) Pax6 constructs used for in vitro translation and GST pull-downs. ( B ) GST pull-down assays with Pax6 HD and PD fused to GST and immobilized on glutathione–agarose beads and Pax6ΔPDΔHD, Pax6ΔPDΔh2–3 or Pax6ΔPDΔh3 produced by in vitro transcription and translation in the presence of [ 35 S]methionine. An aliquot of 10 μl of the in vitro translation reactions was preincubated with GST immobilized on glutathione–agarose beads before incubation with the GST fusion proteins. The GST beads, GST-Pax6 HD beads and GST-Pax6 PD beads were washed several times before they were boiled in SDS loading buffer and run on a 10% SDS–polyacrylamide gel. An aliquot of 2 μl of the in vitro translated proteins was run on the same gel to visualize the signal from 20% of the input. ( C ) Point mutations in helix 3 of the homeodomain strongly reduce the ability of Pax6 HD to interact with the PD and the wild-type HD. The N51Q, R53A and R58A, but not S50A, mutants impede the HD–PD and HD–HD interactions. GST pull-down assays were performed with recombinant GST fusions of wild type or mutants of Pax6ΔHD against in vitro translated, [ 35 S]methionine-labeled Pax6, Pax6ΔHD or Pax6ΔPD. ( D ) The interactions between full-length Pax6 and the RED subdomain and between full-length Pax6 and the HD are independent of DNA. GST pull-down assays were done with Pax6 HD and RED fused to GST as in (C). Where indicated, the pull-down experiments were performed in the presence of 500 U benzonase to degrade both DNA and RNA. The results shown are representative of three independent experiments. ( E ) The PD–HD and HD–HD interactions of Pax6 are also observed in the yeast-based SOS recruitment interaction system. The temperature sensitive yeast strain S.cerevisiae cdc25-2 MATa was co-transformed either with pSOS-zfPax6-HDwt and empty pMYR or pMYR-LaminC as negative controls, pMYR-SOS binding protein as a positive control, pMYR-zfPax6-HDwt, or with pMYR-zfPax6-PDwt. Three independent colonies generated from each co-transformation were replica plated onto galactose plates and grown in parallel at 25 and 37°C for 6 days. The results shown are representative of three independent experiments.

Techniques Used: Construct, In Vitro, Produced, Incubation, Recombinant, Labeling, Transformation Assay, Binding Assay, Positive Control, Generated

Reduced superactivation of the paired domain mutant Pax6ΔHD(E112A/E120A/E128A) by the paired-class homeodomain protein Chx10. ( A ) The Pax6ΔHD(3E/A) triple mutant show reduced binding to the homeodomain of Chx10. GST pull-down assays with the HD of murine Chx10 fused to GST and immobilized on glutathione–agarose beads and Pax6ΔHD and Pax6ΔHD(3E/A) protein produced by in vitro transcription and translation in the presence of [ 35 S]methionine. The GST pull-downs were performed as described in the legend to Figure 1 . ( B ) NIH 3T3 cells were co-transfected with 5 ng of either pcDNA3-HA vector, HA-Pax6ΔHD or HA-Pax6ΔHD(3E/A) expression vectors together with vector control or increasing amounts of HA-Chx10 expression vector (5, 25 and 100 ng). An aliquot of 50 ng of the pP6CON-LUC reporter vector and 5 ng of the CMV βgal vector were used. The data are shown as fold superactivation compared with Pax6ΔHD and empty vector control. The data are representative of two other independent experiments.
Figure Legend Snippet: Reduced superactivation of the paired domain mutant Pax6ΔHD(E112A/E120A/E128A) by the paired-class homeodomain protein Chx10. ( A ) The Pax6ΔHD(3E/A) triple mutant show reduced binding to the homeodomain of Chx10. GST pull-down assays with the HD of murine Chx10 fused to GST and immobilized on glutathione–agarose beads and Pax6ΔHD and Pax6ΔHD(3E/A) protein produced by in vitro transcription and translation in the presence of [ 35 S]methionine. The GST pull-downs were performed as described in the legend to Figure 1 . ( B ) NIH 3T3 cells were co-transfected with 5 ng of either pcDNA3-HA vector, HA-Pax6ΔHD or HA-Pax6ΔHD(3E/A) expression vectors together with vector control or increasing amounts of HA-Chx10 expression vector (5, 25 and 100 ng). An aliquot of 50 ng of the pP6CON-LUC reporter vector and 5 ng of the CMV βgal vector were used. The data are shown as fold superactivation compared with Pax6ΔHD and empty vector control. The data are representative of two other independent experiments.

Techniques Used: Mutagenesis, Binding Assay, Produced, In Vitro, Transfection, Plasmid Preparation, Expressing

14) Product Images from "Herpes Simplex Virus ICP27 Protein Directly Interacts with the Nuclear Pore Complex through Nup62, Inhibiting Host Nucleocytoplasmic Transport Pathways *"

Article Title: Herpes Simplex Virus ICP27 Protein Directly Interacts with the Nuclear Pore Complex through Nup62, Inhibiting Host Nucleocytoplasmic Transport Pathways *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.331777

Interaction of GST-Nup62 and ICP27 does not depend on RNA or virus infection. A , Nup62 fused to GST and purified from bacteria in the absence of any other mammalian proteins. Nup62-GST was incubated with WT HSV-1-, Δ27-, and mock-infected cell extracts or with extracts from uninfected cells expressing ICP27 from a plasmid behind the CMV promoter. Upper panel , Western blotting with Abs against ICP27. Lower panel , Western blotting with GAPDH Abs as a loading control for lysates. Input and pull-downs with GST alone were added on the ends for controls. B , GST-Nup62 was incubated with WT HSV-1-infected lysates either with or without RNase I treatment. Again, GST alone and input were used as controls. C , Rae1, another transport receptor, does not pull down ICP27. Bacterially purified GST-Rae1 (67 kDa) was incubated with lysates from WT HSV-1-, Δ27 null virus-, a mutant HSV-1 strain (M15) carrying a C-terminal point mutant (P465L/G466E) in ICP27-, or mock-infected cells. Upper panel , proteins co-purifying on glutathione-Sepharose beads were analyzed by Western blotting with Ab against ICP27. GST-Rae1 did not pull down ICP27 from any of the added cell extracts. A doublet band running lower in lanes 2–5 and marked with an asterisk is a band from the GST-Rae1 bacterial protein preparation cross-reacting nonspecifically with anti-ICP27 Ab. D , bacterially expressed GST-Rae1 protein from the fusion protein preparation used in binding assays shown in Fig. 4 C was analyzed on a Coomassie-stained gel. The expected full-length protein position is shown with an asterisk , whereas the double asterisks indicate cleavage products of the full-length protein. E , GST-Rae1 and GST-TAP pull-down known partner protein Nup98. Bacterially purified fusion protein GST-Rae1 (67 kDa) was incubated in the presence of RNase I with HSV-1 Δ27 null virus-infected or mock-infected cell lysates. Another fusion protein, GST-TAP, earlier shown to interact with Nup98, and GST alone were also incubated with HSV-1 WT virus-infected cell lysates. Proteins co-purifying on glutathione-Sepharose beads shown as bound samples and unbound leftover samples were analyzed by Western blotting with Ab against Nup98. Mock-infected and Δ27 null virus-infected cell lysates were loaded as input. Bound samples show that GST-Rae1 pulled down Nup98 from Δ27 null virus-infected or mock-infected cell lysates, and GST-TAP also pulled down Nup98 from WT virus-infected lysates. GST alone showed a weak band as background in bound samples; however, the majority of Nup98 is not pulled down and is present in unbound samples with GST alone, whereas not much is left as unbound with mock.
Figure Legend Snippet: Interaction of GST-Nup62 and ICP27 does not depend on RNA or virus infection. A , Nup62 fused to GST and purified from bacteria in the absence of any other mammalian proteins. Nup62-GST was incubated with WT HSV-1-, Δ27-, and mock-infected cell extracts or with extracts from uninfected cells expressing ICP27 from a plasmid behind the CMV promoter. Upper panel , Western blotting with Abs against ICP27. Lower panel , Western blotting with GAPDH Abs as a loading control for lysates. Input and pull-downs with GST alone were added on the ends for controls. B , GST-Nup62 was incubated with WT HSV-1-infected lysates either with or without RNase I treatment. Again, GST alone and input were used as controls. C , Rae1, another transport receptor, does not pull down ICP27. Bacterially purified GST-Rae1 (67 kDa) was incubated with lysates from WT HSV-1-, Δ27 null virus-, a mutant HSV-1 strain (M15) carrying a C-terminal point mutant (P465L/G466E) in ICP27-, or mock-infected cells. Upper panel , proteins co-purifying on glutathione-Sepharose beads were analyzed by Western blotting with Ab against ICP27. GST-Rae1 did not pull down ICP27 from any of the added cell extracts. A doublet band running lower in lanes 2–5 and marked with an asterisk is a band from the GST-Rae1 bacterial protein preparation cross-reacting nonspecifically with anti-ICP27 Ab. D , bacterially expressed GST-Rae1 protein from the fusion protein preparation used in binding assays shown in Fig. 4 C was analyzed on a Coomassie-stained gel. The expected full-length protein position is shown with an asterisk , whereas the double asterisks indicate cleavage products of the full-length protein. E , GST-Rae1 and GST-TAP pull-down known partner protein Nup98. Bacterially purified fusion protein GST-Rae1 (67 kDa) was incubated in the presence of RNase I with HSV-1 Δ27 null virus-infected or mock-infected cell lysates. Another fusion protein, GST-TAP, earlier shown to interact with Nup98, and GST alone were also incubated with HSV-1 WT virus-infected cell lysates. Proteins co-purifying on glutathione-Sepharose beads shown as bound samples and unbound leftover samples were analyzed by Western blotting with Ab against Nup98. Mock-infected and Δ27 null virus-infected cell lysates were loaded as input. Bound samples show that GST-Rae1 pulled down Nup98 from Δ27 null virus-infected or mock-infected cell lysates, and GST-TAP also pulled down Nup98 from WT virus-infected lysates. GST alone showed a weak band as background in bound samples; however, the majority of Nup98 is not pulled down and is present in unbound samples with GST alone, whereas not much is left as unbound with mock.

Techniques Used: Infection, Purification, Incubation, Expressing, Plasmid Preparation, Western Blot, Mutagenesis, Binding Assay, Staining

The Nup62-ICP27 interaction is direct, and TAP also binds ICP27 under similar conditions. A , GST-TAP pulls down ICP27 from WT HSV-1-infected cell extracts but not from extracts of cells infected with a viral ICP27 C-terminal point mutant (M15; upper panel ), whereas in the same binding reactions, GST-TAP pulls down Nup62 from WT HSV-1-infected cell extracts and from extracts of cells infected with a viral ICP27 C-terminal point mutant (M15) or with ICP27 null mutant Δ27 virus and from mock-infected cell extracts ( upper panel ). Bacterially expressed GST or GST-TAP was incubated with extracts from WT-, Δ27-, or M15-infected HSV-1 strains. Proteins co-purifying on glutathione-Sepharose beads were analyzed by Western blot with ICP27 or Nup62 Abs. B , ICP27 and TAP co-immunoprecipitate from HeLa cell extracts in the presence of RNase I. Co-immunoprecipitation was carried out by mixing anti-ICP27 antibodies 1113 and 1119 with HSV-1 WT and ICP27 mutant M15 and null Δ27 viruses and mock-infected HeLa cell extracts. Complexes formed were separated by SDS-PAGE followed by Western blotting with anti-TAP and ICP27 Abs. ICP27 co-precipitated TAP. Upper panel , co-immunoprecipitation of TAP in WT-infected HeLa cell extracts in the presence of RNase I by ICP27 but no co-immunoprecipitation with M15-, Δ27-, and mock-infected cells. Input , mock-infected HeLa cell extract. The lower band in the input lane could be due to another cellular protein cross-reacting with this TAP Ab, but the correct size upper band specific to TAP is clearly seen. The band marked with an asterisk is the heavy chain of IgGs used for immunoprecipitations. ICP27 immunoprecipitated itself. The lower panel shows immunoprecipitation of ICP27 in WT- and M15-infected HeLa cell extracts in the presence of RNase I by its Ab but no immunoprecipitation in Δ27- and mock-infected cell extracts. C , the purity of various fusion proteins used for in vitro binding assays. Coomassie-stained 10% SDS-PAGE of bacterially expressed proteins used in Figs. 4 – 7 . The single asterisk indicates the molecular weight of the expected protein product, whereas the double asterisks indicate cleavage products of the full-length protein. Prestained molecular weight protein markers are loaded on the leftmost lane with GST alone. D , bacterially purified His-tagged ICP27 was incubated with bacterially purified GST-Nup62, GST, or GST-TAP proteins on glutathione-Sepharose beads. Protein complexes formed were eluted by reduced glutathione and separated by SDS-PAGE and Western blotted with anti-His Ab. His-ICP27 interacted with GST-Nup62 and GST-TAP but not GST alone. E , an unrelated nonspecific His-tagged fusion protein (His-mRFP) does not interact with GST-tagged TAP or Nup62 fusion proteins. Bacterially purified His-tagged mRFP fusion protein was incubated with bacterially purified GST-TAP, GST-Nup62, or GST alone on glutathione-Sepharose beads in the presence of RNase I. Protein complexes eluted by reduced glutathione and separated by SDS-PAGE are shown on a Coomassie-stained gel. His-mRFP (corresponding to the band in input) does not interact with GST-TAP, GST-Nup62, and GST alone in bound samples run on gel ( upper panel ), but His-mRFP is present in all unbound pull-down samples run on Coomassie ( lower panel ). Purified His-mRFP alone protein added to the pull-down reactions was loaded as input. Molecular weight protein marker sizes are given in kDa on the left .
Figure Legend Snippet: The Nup62-ICP27 interaction is direct, and TAP also binds ICP27 under similar conditions. A , GST-TAP pulls down ICP27 from WT HSV-1-infected cell extracts but not from extracts of cells infected with a viral ICP27 C-terminal point mutant (M15; upper panel ), whereas in the same binding reactions, GST-TAP pulls down Nup62 from WT HSV-1-infected cell extracts and from extracts of cells infected with a viral ICP27 C-terminal point mutant (M15) or with ICP27 null mutant Δ27 virus and from mock-infected cell extracts ( upper panel ). Bacterially expressed GST or GST-TAP was incubated with extracts from WT-, Δ27-, or M15-infected HSV-1 strains. Proteins co-purifying on glutathione-Sepharose beads were analyzed by Western blot with ICP27 or Nup62 Abs. B , ICP27 and TAP co-immunoprecipitate from HeLa cell extracts in the presence of RNase I. Co-immunoprecipitation was carried out by mixing anti-ICP27 antibodies 1113 and 1119 with HSV-1 WT and ICP27 mutant M15 and null Δ27 viruses and mock-infected HeLa cell extracts. Complexes formed were separated by SDS-PAGE followed by Western blotting with anti-TAP and ICP27 Abs. ICP27 co-precipitated TAP. Upper panel , co-immunoprecipitation of TAP in WT-infected HeLa cell extracts in the presence of RNase I by ICP27 but no co-immunoprecipitation with M15-, Δ27-, and mock-infected cells. Input , mock-infected HeLa cell extract. The lower band in the input lane could be due to another cellular protein cross-reacting with this TAP Ab, but the correct size upper band specific to TAP is clearly seen. The band marked with an asterisk is the heavy chain of IgGs used for immunoprecipitations. ICP27 immunoprecipitated itself. The lower panel shows immunoprecipitation of ICP27 in WT- and M15-infected HeLa cell extracts in the presence of RNase I by its Ab but no immunoprecipitation in Δ27- and mock-infected cell extracts. C , the purity of various fusion proteins used for in vitro binding assays. Coomassie-stained 10% SDS-PAGE of bacterially expressed proteins used in Figs. 4 – 7 . The single asterisk indicates the molecular weight of the expected protein product, whereas the double asterisks indicate cleavage products of the full-length protein. Prestained molecular weight protein markers are loaded on the leftmost lane with GST alone. D , bacterially purified His-tagged ICP27 was incubated with bacterially purified GST-Nup62, GST, or GST-TAP proteins on glutathione-Sepharose beads. Protein complexes formed were eluted by reduced glutathione and separated by SDS-PAGE and Western blotted with anti-His Ab. His-ICP27 interacted with GST-Nup62 and GST-TAP but not GST alone. E , an unrelated nonspecific His-tagged fusion protein (His-mRFP) does not interact with GST-tagged TAP or Nup62 fusion proteins. Bacterially purified His-tagged mRFP fusion protein was incubated with bacterially purified GST-TAP, GST-Nup62, or GST alone on glutathione-Sepharose beads in the presence of RNase I. Protein complexes eluted by reduced glutathione and separated by SDS-PAGE are shown on a Coomassie-stained gel. His-mRFP (corresponding to the band in input) does not interact with GST-TAP, GST-Nup62, and GST alone in bound samples run on gel ( upper panel ), but His-mRFP is present in all unbound pull-down samples run on Coomassie ( lower panel ). Purified His-mRFP alone protein added to the pull-down reactions was loaded as input. Molecular weight protein marker sizes are given in kDa on the left .

Techniques Used: Infection, Mutagenesis, Binding Assay, Incubation, Western Blot, Immunoprecipitation, SDS Page, In Vitro, Staining, Molecular Weight, Purification, Marker

Mapping of Nup62 interaction sites on ICP27. A, schematic of ICP27, N-terminal deletion mutants, and C-terminal point mutants. The NES, NLS, and the RGG viral RNA binding site are marked along with the three hnRNP K homology domains ( KH1 to - 3 ). In the third conserved domain are the Sm and zinc finger regions that are disrupted by the M15 (P465L/G466E) and M16 point mutations (C488L) ( 57 ), respectively, and are represented with double and single asterisks , respectively. B , GST-Nup62 was incubated with lysates from cells infected with herpesviruses carrying the listed ICP27 mutations. The material that bound to GST-Nup62 and eluted from glutathione-Sepharose beads is shown in the upper panels , whereas the unbound material is shown in the lower panels to confirm that the viral proteins were expressed. All co-precipitated proteins are visualized with the ICP27 monoclonal Abs, either 1113 or 1119 because 1113 Ab is raised against a region of ICP27 that is missing in mutant d3–4 and 1119 Ab is raised against a region that is missing in dleu mutant. Standard GST alone and input controls were employed.
Figure Legend Snippet: Mapping of Nup62 interaction sites on ICP27. A, schematic of ICP27, N-terminal deletion mutants, and C-terminal point mutants. The NES, NLS, and the RGG viral RNA binding site are marked along with the three hnRNP K homology domains ( KH1 to - 3 ). In the third conserved domain are the Sm and zinc finger regions that are disrupted by the M15 (P465L/G466E) and M16 point mutations (C488L) ( 57 ), respectively, and are represented with double and single asterisks , respectively. B , GST-Nup62 was incubated with lysates from cells infected with herpesviruses carrying the listed ICP27 mutations. The material that bound to GST-Nup62 and eluted from glutathione-Sepharose beads is shown in the upper panels , whereas the unbound material is shown in the lower panels to confirm that the viral proteins were expressed. All co-precipitated proteins are visualized with the ICP27 monoclonal Abs, either 1113 or 1119 because 1113 Ab is raised against a region of ICP27 that is missing in mutant d3–4 and 1119 Ab is raised against a region that is missing in dleu mutant. Standard GST alone and input controls were employed.

Techniques Used: RNA Binding Assay, Incubation, Infection, Mutagenesis

Nup62 interacts with ORF57, an ICP27 homologue from KSHV. A , mock-infected HeLa extracts were incubated with either GST-ICP27, GST-ORF57 (a homologue of ICP27 from KSHV), or GST alone. Complexes isolated on glutathione-Sepharose beads were eluted after washing, separated on 10% SDS-PAGE, transferred to nitrocellulose membranes, and incubated with Abs against Nup62. Both ICP27 homologs pulled down Nup62. 33% of the HeLa cell extracts used for binding were loaded as input, and one-half of the pull-down reaction samples eluted from the beads were loaded on the gel. The lower band marked with an asterisk in lane 2 is a band from the GST-ICP27 protein preparation cross-reacting nonspecifically with Nup62 Ab. B , control protein GAPDH did not bind to ICP27 and ORF57 fusion proteins. Pulled down reactions of ICP27, ORF57 fusion proteins, and GST alone with HeLa lysates were blotted with anti-GAPDH Ab, and this showed GAPDH present only in the input and not pulled down in bound samples. C , Coomassie-stained 10% SDS-PAGE of bacterially expressed proteins GST-ICP27, GST-ORF57, and GST alone present on the beads used in the binding reactions here and in Fig. 5 A . The single asterisk indicates the molecular weight of the expected protein product, whereas the double asterisks indicate cleavage products of the full-length protein.
Figure Legend Snippet: Nup62 interacts with ORF57, an ICP27 homologue from KSHV. A , mock-infected HeLa extracts were incubated with either GST-ICP27, GST-ORF57 (a homologue of ICP27 from KSHV), or GST alone. Complexes isolated on glutathione-Sepharose beads were eluted after washing, separated on 10% SDS-PAGE, transferred to nitrocellulose membranes, and incubated with Abs against Nup62. Both ICP27 homologs pulled down Nup62. 33% of the HeLa cell extracts used for binding were loaded as input, and one-half of the pull-down reaction samples eluted from the beads were loaded on the gel. The lower band marked with an asterisk in lane 2 is a band from the GST-ICP27 protein preparation cross-reacting nonspecifically with Nup62 Ab. B , control protein GAPDH did not bind to ICP27 and ORF57 fusion proteins. Pulled down reactions of ICP27, ORF57 fusion proteins, and GST alone with HeLa lysates were blotted with anti-GAPDH Ab, and this showed GAPDH present only in the input and not pulled down in bound samples. C , Coomassie-stained 10% SDS-PAGE of bacterially expressed proteins GST-ICP27, GST-ORF57, and GST alone present on the beads used in the binding reactions here and in Fig. 5 A . The single asterisk indicates the molecular weight of the expected protein product, whereas the double asterisks indicate cleavage products of the full-length protein.

Techniques Used: Infection, Incubation, Isolation, SDS Page, Binding Assay, Staining, Molecular Weight

15) Product Images from "The integrin cytoplasmic domain-associated protein ICAP-1 binds and regulates Rho family GTPases during cell spreading"

Article Title: The integrin cytoplasmic domain-associated protein ICAP-1 binds and regulates Rho family GTPases during cell spreading

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200108030

ICAP-1 does neither inhibit nor bind RhoA. (A) RhoA activity assay was performed as described in Materials and methods using COS cells either untransfected or transfected as indicated. The same blot was reprobed for ICAP-1 expression with a polyclonal antibody against ICAP-1. (B) Total protein extract of COS cells was loaded on Sepharose coupled with MBP–ICAP-1 fusion protein or MBP as control. The eluted proteins were analyzed by Western blotting with a monoclonal antibody against RhoA. The same filter was stripped and tested for the presence of Rac1 using a specific monoclonal antibody (B'). Total extract is shown as control.
Figure Legend Snippet: ICAP-1 does neither inhibit nor bind RhoA. (A) RhoA activity assay was performed as described in Materials and methods using COS cells either untransfected or transfected as indicated. The same blot was reprobed for ICAP-1 expression with a polyclonal antibody against ICAP-1. (B) Total protein extract of COS cells was loaded on Sepharose coupled with MBP–ICAP-1 fusion protein or MBP as control. The eluted proteins were analyzed by Western blotting with a monoclonal antibody against RhoA. The same filter was stripped and tested for the presence of Rac1 using a specific monoclonal antibody (B'). Total extract is shown as control.

Techniques Used: Activity Assay, Transfection, Expressing, Western Blot

ICAP-1 inhibits and binds Cdc42. (A) COS cells transiently transfected with myc-Cdc42 together with control vector or myc–ICAP-1 were suspended by trypsin treatment, kept in suspension 2 h 30 min in serum-free DME (Su), and plated on fibronectin (5 μg/ml) for 1 h (Ad). Cdc42 activity assay was performed as described in Materials and methods using a GST-PAK-CD fusion protein that selectively binds to GTP-Cdc42. The bound GTP-Cdc42 was analyzed by Western blotting with a polyclonal antibody to Cdc42. To probe for Cdc42 and ICAP-1 expression, total cell lysates were blotted with the corresponding antibodies. (B) The amount of GTP-bound and total Cdc42 (A, bottom band of the doublets) was quantified by densitometric analysis, and the activation level of Cdc42 was expressed as a ratio between the values of GTP-bound and total Cdc42. Comparable results were obtained in three independent experiments. (C) Total protein extract of COS cells transfected with GST-Cdc42 was loaded on Sepharose coupled with MBP–ICAP-1 fusion protein or MBP as control. The proteins eluted with glycine-HCl, pH 3.0, buffer (fractions numbers 1-2-3-4) were analyzed by Western blotting with polyclonal antibody against Cdc42. Note that in A, Cdc42 is detected as a doublet of bands; comparison of the electrophoretic mobility indicates that the bottom band of the doublet in A comigrates with the band in C. The top band of the doublet is likely to represent an incompletely processed form of the GTPase. (D) Western blotting with an antibody against Cdc42 of the pooled fractions eluted from MBP–ICAP-1-Sepharose columns (right). The columns were loaded with equal amounts of GST-Cdc42 fusion protein obtained from either the membrane fraction of pCEFL-GST-Cdc42–transfected COS cells (a), the cytoplasmic fraction of the same cells (b), or bacterial lysate of E. coli producing the GST-Cdc42 fusion protein (c). The amount of fusion proteins loaded on the column are shown on the left as control for equal loading.
Figure Legend Snippet: ICAP-1 inhibits and binds Cdc42. (A) COS cells transiently transfected with myc-Cdc42 together with control vector or myc–ICAP-1 were suspended by trypsin treatment, kept in suspension 2 h 30 min in serum-free DME (Su), and plated on fibronectin (5 μg/ml) for 1 h (Ad). Cdc42 activity assay was performed as described in Materials and methods using a GST-PAK-CD fusion protein that selectively binds to GTP-Cdc42. The bound GTP-Cdc42 was analyzed by Western blotting with a polyclonal antibody to Cdc42. To probe for Cdc42 and ICAP-1 expression, total cell lysates were blotted with the corresponding antibodies. (B) The amount of GTP-bound and total Cdc42 (A, bottom band of the doublets) was quantified by densitometric analysis, and the activation level of Cdc42 was expressed as a ratio between the values of GTP-bound and total Cdc42. Comparable results were obtained in three independent experiments. (C) Total protein extract of COS cells transfected with GST-Cdc42 was loaded on Sepharose coupled with MBP–ICAP-1 fusion protein or MBP as control. The proteins eluted with glycine-HCl, pH 3.0, buffer (fractions numbers 1-2-3-4) were analyzed by Western blotting with polyclonal antibody against Cdc42. Note that in A, Cdc42 is detected as a doublet of bands; comparison of the electrophoretic mobility indicates that the bottom band of the doublet in A comigrates with the band in C. The top band of the doublet is likely to represent an incompletely processed form of the GTPase. (D) Western blotting with an antibody against Cdc42 of the pooled fractions eluted from MBP–ICAP-1-Sepharose columns (right). The columns were loaded with equal amounts of GST-Cdc42 fusion protein obtained from either the membrane fraction of pCEFL-GST-Cdc42–transfected COS cells (a), the cytoplasmic fraction of the same cells (b), or bacterial lysate of E. coli producing the GST-Cdc42 fusion protein (c). The amount of fusion proteins loaded on the column are shown on the left as control for equal loading.

Techniques Used: Transfection, Plasmid Preparation, Activity Assay, Western Blot, Expressing, Activation Assay

ICAP-1 inhibits and binds Rac1. (A) COS cells transiently transfected with control vector or myc–ICAP-1 were suspended by trypsin treatment, kept in suspension 2 h 30 min in serum-free DME (Su), and plated on fibronectin (5 μg/ml) for 1 h (Ad). Rac1 activation level was evaluated as described in Materials and methods using a GST-PAK-CD fusion protein that selectively binds to GTP-Rac1. The bound GTP-Rac1 was analyzed by Western blotting with a polyclonal antibody to Rac1. To probe for Rac1 and ICAP-1 expression, total cell lysates were blotted with the corresponding antibodies. (B) The amount of GTP-bound and total Rac1 (A) was quantified by densitometric analysis; the activation level of Rac1 is expressed as a ratio between the values of GTP-bound and total Rac1. Comparable results were obtained in three independent experiments. (C) Total lysate of COS cells transfected with myc-Rac1 was loaded on Sepharose coupled with MBP–ICAP-1 fusion protein or MBP as control. The proteins eluted with glycine-HCl, pH 3, buffer (fractions numbers 1-2-3-4) were analyzed by Western blotting with a monoclonal antibody against Rac1.
Figure Legend Snippet: ICAP-1 inhibits and binds Rac1. (A) COS cells transiently transfected with control vector or myc–ICAP-1 were suspended by trypsin treatment, kept in suspension 2 h 30 min in serum-free DME (Su), and plated on fibronectin (5 μg/ml) for 1 h (Ad). Rac1 activation level was evaluated as described in Materials and methods using a GST-PAK-CD fusion protein that selectively binds to GTP-Rac1. The bound GTP-Rac1 was analyzed by Western blotting with a polyclonal antibody to Rac1. To probe for Rac1 and ICAP-1 expression, total cell lysates were blotted with the corresponding antibodies. (B) The amount of GTP-bound and total Rac1 (A) was quantified by densitometric analysis; the activation level of Rac1 is expressed as a ratio between the values of GTP-bound and total Rac1. Comparable results were obtained in three independent experiments. (C) Total lysate of COS cells transfected with myc-Rac1 was loaded on Sepharose coupled with MBP–ICAP-1 fusion protein or MBP as control. The proteins eluted with glycine-HCl, pH 3, buffer (fractions numbers 1-2-3-4) were analyzed by Western blotting with a monoclonal antibody against Rac1.

Techniques Used: Transfection, Plasmid Preparation, Activation Assay, Western Blot, Expressing

Inhibitory effect of ICAP-1 on the dissociation of [ α - 32 P]GDP from Cdc42. GST-Cdc42 was expressed in COS cells, and the protein was purified from the membranous fraction and captured on glutathione-Sepharose. The Cdc42 immobilized on the beads was loaded with guanosine nucleotide by incubation with [α- 32 P]GTP 25 min at room temperature. The dissociation of the GDP induced by chelating Mg 2+ with EDTA (A) or by the GEF activity of Dbl (B) was measured in the presence of either the buffer alone, MBP–ICAP-1(10 μg), or the same amount of GST-Rho GDI fusion proteins after 10 min at room temperature. (C) Time course for [α- 32 P]GDP dissociation from Cdc42 induced by chelating Mg 2+ with EDTA in the presence of either 10 μg of MBP–ICAP-1, GST-Rho GDI, MBP carrier protein, or buffer alone. Results shown are means ± SD of values from three experiments. [α- 32 P]GDP remaining is expressed as the percentage of [α- 32 P]GDP bound to Cdc42 after loading.
Figure Legend Snippet: Inhibitory effect of ICAP-1 on the dissociation of [ α - 32 P]GDP from Cdc42. GST-Cdc42 was expressed in COS cells, and the protein was purified from the membranous fraction and captured on glutathione-Sepharose. The Cdc42 immobilized on the beads was loaded with guanosine nucleotide by incubation with [α- 32 P]GTP 25 min at room temperature. The dissociation of the GDP induced by chelating Mg 2+ with EDTA (A) or by the GEF activity of Dbl (B) was measured in the presence of either the buffer alone, MBP–ICAP-1(10 μg), or the same amount of GST-Rho GDI fusion proteins after 10 min at room temperature. (C) Time course for [α- 32 P]GDP dissociation from Cdc42 induced by chelating Mg 2+ with EDTA in the presence of either 10 μg of MBP–ICAP-1, GST-Rho GDI, MBP carrier protein, or buffer alone. Results shown are means ± SD of values from three experiments. [α- 32 P]GDP remaining is expressed as the percentage of [α- 32 P]GDP bound to Cdc42 after loading.

Techniques Used: Purification, Incubation, Activity Assay

16) Product Images from "Herpes Simplex Virus ICP27 Protein Directly Interacts with the Nuclear Pore Complex through Nup62, Inhibiting Host Nucleocytoplasmic Transport Pathways *"

Article Title: Herpes Simplex Virus ICP27 Protein Directly Interacts with the Nuclear Pore Complex through Nup62, Inhibiting Host Nucleocytoplasmic Transport Pathways *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.331777

Interaction of GST-Nup62 and ICP27 does not depend on RNA or virus infection. A , Nup62 fused to GST and purified from bacteria in the absence of any other mammalian proteins. Nup62-GST was incubated with WT HSV-1-, Δ27-, and mock-infected cell extracts or with extracts from uninfected cells expressing ICP27 from a plasmid behind the CMV promoter. Upper panel , Western blotting with Abs against ICP27. Lower panel , Western blotting with GAPDH Abs as a loading control for lysates. Input and pull-downs with GST alone were added on the ends for controls. B , GST-Nup62 was incubated with WT HSV-1-infected lysates either with or without RNase I treatment. Again, GST alone and input were used as controls. C , Rae1, another transport receptor, does not pull down ICP27. Bacterially purified GST-Rae1 (67 kDa) was incubated with lysates from WT HSV-1-, Δ27 null virus-, a mutant HSV-1 strain (M15) carrying a C-terminal point mutant (P465L/G466E) in ICP27-, or mock-infected cells. Upper panel , proteins co-purifying on glutathione-Sepharose beads were analyzed by Western blotting with Ab against ICP27. GST-Rae1 did not pull down ICP27 from any of the added cell extracts. A doublet band running lower in lanes 2–5 and marked with an asterisk is a band from the GST-Rae1 bacterial protein preparation cross-reacting nonspecifically with anti-ICP27 Ab. D , bacterially expressed GST-Rae1 protein from the fusion protein preparation used in binding assays shown in Fig. 4 C was analyzed on a Coomassie-stained gel. The expected full-length protein position is shown with an asterisk , whereas the double asterisks indicate cleavage products of the full-length protein. E , GST-Rae1 and GST-TAP pull-down known partner protein Nup98. Bacterially purified fusion protein GST-Rae1 (67 kDa) was incubated in the presence of RNase I with HSV-1 Δ27 null virus-infected or mock-infected cell lysates. Another fusion protein, GST-TAP, earlier shown to interact with Nup98, and GST alone were also incubated with HSV-1 WT virus-infected cell lysates. Proteins co-purifying on glutathione-Sepharose beads shown as bound samples and unbound leftover samples were analyzed by Western blotting with Ab against Nup98. Mock-infected and Δ27 null virus-infected cell lysates were loaded as input. Bound samples show that GST-Rae1 pulled down Nup98 from Δ27 null virus-infected or mock-infected cell lysates, and GST-TAP also pulled down Nup98 from WT virus-infected lysates. GST alone showed a weak band as background in bound samples; however, the majority of Nup98 is not pulled down and is present in unbound samples with GST alone, whereas not much is left as unbound with mock.
Figure Legend Snippet: Interaction of GST-Nup62 and ICP27 does not depend on RNA or virus infection. A , Nup62 fused to GST and purified from bacteria in the absence of any other mammalian proteins. Nup62-GST was incubated with WT HSV-1-, Δ27-, and mock-infected cell extracts or with extracts from uninfected cells expressing ICP27 from a plasmid behind the CMV promoter. Upper panel , Western blotting with Abs against ICP27. Lower panel , Western blotting with GAPDH Abs as a loading control for lysates. Input and pull-downs with GST alone were added on the ends for controls. B , GST-Nup62 was incubated with WT HSV-1-infected lysates either with or without RNase I treatment. Again, GST alone and input were used as controls. C , Rae1, another transport receptor, does not pull down ICP27. Bacterially purified GST-Rae1 (67 kDa) was incubated with lysates from WT HSV-1-, Δ27 null virus-, a mutant HSV-1 strain (M15) carrying a C-terminal point mutant (P465L/G466E) in ICP27-, or mock-infected cells. Upper panel , proteins co-purifying on glutathione-Sepharose beads were analyzed by Western blotting with Ab against ICP27. GST-Rae1 did not pull down ICP27 from any of the added cell extracts. A doublet band running lower in lanes 2–5 and marked with an asterisk is a band from the GST-Rae1 bacterial protein preparation cross-reacting nonspecifically with anti-ICP27 Ab. D , bacterially expressed GST-Rae1 protein from the fusion protein preparation used in binding assays shown in Fig. 4 C was analyzed on a Coomassie-stained gel. The expected full-length protein position is shown with an asterisk , whereas the double asterisks indicate cleavage products of the full-length protein. E , GST-Rae1 and GST-TAP pull-down known partner protein Nup98. Bacterially purified fusion protein GST-Rae1 (67 kDa) was incubated in the presence of RNase I with HSV-1 Δ27 null virus-infected or mock-infected cell lysates. Another fusion protein, GST-TAP, earlier shown to interact with Nup98, and GST alone were also incubated with HSV-1 WT virus-infected cell lysates. Proteins co-purifying on glutathione-Sepharose beads shown as bound samples and unbound leftover samples were analyzed by Western blotting with Ab against Nup98. Mock-infected and Δ27 null virus-infected cell lysates were loaded as input. Bound samples show that GST-Rae1 pulled down Nup98 from Δ27 null virus-infected or mock-infected cell lysates, and GST-TAP also pulled down Nup98 from WT virus-infected lysates. GST alone showed a weak band as background in bound samples; however, the majority of Nup98 is not pulled down and is present in unbound samples with GST alone, whereas not much is left as unbound with mock.

Techniques Used: Infection, Purification, Incubation, Expressing, Plasmid Preparation, Western Blot, Mutagenesis, Binding Assay, Staining

The Nup62-ICP27 interaction is direct, and TAP also binds ICP27 under similar conditions. A , GST-TAP pulls down ICP27 from WT HSV-1-infected cell extracts but not from extracts of cells infected with a viral ICP27 C-terminal point mutant (M15; upper panel ), whereas in the same binding reactions, GST-TAP pulls down Nup62 from WT HSV-1-infected cell extracts and from extracts of cells infected with a viral ICP27 C-terminal point mutant (M15) or with ICP27 null mutant Δ27 virus and from mock-infected cell extracts ( upper panel ). Bacterially expressed GST or GST-TAP was incubated with extracts from WT-, Δ27-, or M15-infected HSV-1 strains. Proteins co-purifying on glutathione-Sepharose beads were analyzed by Western blot with ICP27 or Nup62 Abs. B , ICP27 and TAP co-immunoprecipitate from HeLa cell extracts in the presence of RNase I. Co-immunoprecipitation was carried out by mixing anti-ICP27 antibodies 1113 and 1119 with HSV-1 WT and ICP27 mutant M15 and null Δ27 viruses and mock-infected HeLa cell extracts. Complexes formed were separated by SDS-PAGE followed by Western blotting with anti-TAP and ICP27 Abs. ICP27 co-precipitated TAP. Upper panel , co-immunoprecipitation of TAP in WT-infected HeLa cell extracts in the presence of RNase I by ICP27 but no co-immunoprecipitation with M15-, Δ27-, and mock-infected cells. Input , mock-infected HeLa cell extract. The lower band in the input lane could be due to another cellular protein cross-reacting with this TAP Ab, but the correct size upper band specific to TAP is clearly seen. The band marked with an asterisk is the heavy chain of IgGs used for immunoprecipitations. ICP27 immunoprecipitated itself. The lower panel shows immunoprecipitation of ICP27 in WT- and M15-infected HeLa cell extracts in the presence of RNase I by its Ab but no immunoprecipitation in Δ27- and mock-infected cell extracts. C , the purity of various fusion proteins used for in vitro binding assays. Coomassie-stained 10% SDS-PAGE of bacterially expressed proteins used in Figs. 4 – 7 . The single asterisk indicates the molecular weight of the expected protein product, whereas the double asterisks indicate cleavage products of the full-length protein. Prestained molecular weight protein markers are loaded on the leftmost lane with GST alone. D , bacterially purified His-tagged ICP27 was incubated with bacterially purified GST-Nup62, GST, or GST-TAP proteins on glutathione-Sepharose beads. Protein complexes formed were eluted by reduced glutathione and separated by SDS-PAGE and Western blotted with anti-His Ab. His-ICP27 interacted with GST-Nup62 and GST-TAP but not GST alone. E , an unrelated nonspecific His-tagged fusion protein (His-mRFP) does not interact with GST-tagged TAP or Nup62 fusion proteins. Bacterially purified His-tagged mRFP fusion protein was incubated with bacterially purified GST-TAP, GST-Nup62, or GST alone on glutathione-Sepharose beads in the presence of RNase I. Protein complexes eluted by reduced glutathione and separated by SDS-PAGE are shown on a Coomassie-stained gel. His-mRFP (corresponding to the band in input) does not interact with GST-TAP, GST-Nup62, and GST alone in bound samples run on gel ( upper panel ), but His-mRFP is present in all unbound pull-down samples run on Coomassie ( lower panel ). Purified His-mRFP alone protein added to the pull-down reactions was loaded as input. Molecular weight protein marker sizes are given in kDa on the left .
Figure Legend Snippet: The Nup62-ICP27 interaction is direct, and TAP also binds ICP27 under similar conditions. A , GST-TAP pulls down ICP27 from WT HSV-1-infected cell extracts but not from extracts of cells infected with a viral ICP27 C-terminal point mutant (M15; upper panel ), whereas in the same binding reactions, GST-TAP pulls down Nup62 from WT HSV-1-infected cell extracts and from extracts of cells infected with a viral ICP27 C-terminal point mutant (M15) or with ICP27 null mutant Δ27 virus and from mock-infected cell extracts ( upper panel ). Bacterially expressed GST or GST-TAP was incubated with extracts from WT-, Δ27-, or M15-infected HSV-1 strains. Proteins co-purifying on glutathione-Sepharose beads were analyzed by Western blot with ICP27 or Nup62 Abs. B , ICP27 and TAP co-immunoprecipitate from HeLa cell extracts in the presence of RNase I. Co-immunoprecipitation was carried out by mixing anti-ICP27 antibodies 1113 and 1119 with HSV-1 WT and ICP27 mutant M15 and null Δ27 viruses and mock-infected HeLa cell extracts. Complexes formed were separated by SDS-PAGE followed by Western blotting with anti-TAP and ICP27 Abs. ICP27 co-precipitated TAP. Upper panel , co-immunoprecipitation of TAP in WT-infected HeLa cell extracts in the presence of RNase I by ICP27 but no co-immunoprecipitation with M15-, Δ27-, and mock-infected cells. Input , mock-infected HeLa cell extract. The lower band in the input lane could be due to another cellular protein cross-reacting with this TAP Ab, but the correct size upper band specific to TAP is clearly seen. The band marked with an asterisk is the heavy chain of IgGs used for immunoprecipitations. ICP27 immunoprecipitated itself. The lower panel shows immunoprecipitation of ICP27 in WT- and M15-infected HeLa cell extracts in the presence of RNase I by its Ab but no immunoprecipitation in Δ27- and mock-infected cell extracts. C , the purity of various fusion proteins used for in vitro binding assays. Coomassie-stained 10% SDS-PAGE of bacterially expressed proteins used in Figs. 4 – 7 . The single asterisk indicates the molecular weight of the expected protein product, whereas the double asterisks indicate cleavage products of the full-length protein. Prestained molecular weight protein markers are loaded on the leftmost lane with GST alone. D , bacterially purified His-tagged ICP27 was incubated with bacterially purified GST-Nup62, GST, or GST-TAP proteins on glutathione-Sepharose beads. Protein complexes formed were eluted by reduced glutathione and separated by SDS-PAGE and Western blotted with anti-His Ab. His-ICP27 interacted with GST-Nup62 and GST-TAP but not GST alone. E , an unrelated nonspecific His-tagged fusion protein (His-mRFP) does not interact with GST-tagged TAP or Nup62 fusion proteins. Bacterially purified His-tagged mRFP fusion protein was incubated with bacterially purified GST-TAP, GST-Nup62, or GST alone on glutathione-Sepharose beads in the presence of RNase I. Protein complexes eluted by reduced glutathione and separated by SDS-PAGE are shown on a Coomassie-stained gel. His-mRFP (corresponding to the band in input) does not interact with GST-TAP, GST-Nup62, and GST alone in bound samples run on gel ( upper panel ), but His-mRFP is present in all unbound pull-down samples run on Coomassie ( lower panel ). Purified His-mRFP alone protein added to the pull-down reactions was loaded as input. Molecular weight protein marker sizes are given in kDa on the left .

Techniques Used: Infection, Mutagenesis, Binding Assay, Incubation, Western Blot, Immunoprecipitation, SDS Page, In Vitro, Staining, Molecular Weight, Purification, Marker

Mapping of Nup62 interaction sites on ICP27. A, schematic of ICP27, N-terminal deletion mutants, and C-terminal point mutants. The NES, NLS, and the RGG viral RNA binding site are marked along with the three hnRNP K homology domains ( KH1 to - 3 ). In the third conserved domain are the Sm and zinc finger regions that are disrupted by the M15 (P465L/G466E) and M16 point mutations (C488L) ( 57 ), respectively, and are represented with double and single asterisks , respectively. B , GST-Nup62 was incubated with lysates from cells infected with herpesviruses carrying the listed ICP27 mutations. The material that bound to GST-Nup62 and eluted from glutathione-Sepharose beads is shown in the upper panels , whereas the unbound material is shown in the lower panels to confirm that the viral proteins were expressed. All co-precipitated proteins are visualized with the ICP27 monoclonal Abs, either 1113 or 1119 because 1113 Ab is raised against a region of ICP27 that is missing in mutant d3–4 and 1119 Ab is raised against a region that is missing in dleu mutant. Standard GST alone and input controls were employed.
Figure Legend Snippet: Mapping of Nup62 interaction sites on ICP27. A, schematic of ICP27, N-terminal deletion mutants, and C-terminal point mutants. The NES, NLS, and the RGG viral RNA binding site are marked along with the three hnRNP K homology domains ( KH1 to - 3 ). In the third conserved domain are the Sm and zinc finger regions that are disrupted by the M15 (P465L/G466E) and M16 point mutations (C488L) ( 57 ), respectively, and are represented with double and single asterisks , respectively. B , GST-Nup62 was incubated with lysates from cells infected with herpesviruses carrying the listed ICP27 mutations. The material that bound to GST-Nup62 and eluted from glutathione-Sepharose beads is shown in the upper panels , whereas the unbound material is shown in the lower panels to confirm that the viral proteins were expressed. All co-precipitated proteins are visualized with the ICP27 monoclonal Abs, either 1113 or 1119 because 1113 Ab is raised against a region of ICP27 that is missing in mutant d3–4 and 1119 Ab is raised against a region that is missing in dleu mutant. Standard GST alone and input controls were employed.

Techniques Used: RNA Binding Assay, Incubation, Infection, Mutagenesis

Nup62 interacts with ORF57, an ICP27 homologue from KSHV. A , mock-infected HeLa extracts were incubated with either GST-ICP27, GST-ORF57 (a homologue of ICP27 from KSHV), or GST alone. Complexes isolated on glutathione-Sepharose beads were eluted after washing, separated on 10% SDS-PAGE, transferred to nitrocellulose membranes, and incubated with Abs against Nup62. Both ICP27 homologs pulled down Nup62. 33% of the HeLa cell extracts used for binding were loaded as input, and one-half of the pull-down reaction samples eluted from the beads were loaded on the gel. The lower band marked with an asterisk in lane 2 is a band from the GST-ICP27 protein preparation cross-reacting nonspecifically with Nup62 Ab. B , control protein GAPDH did not bind to ICP27 and ORF57 fusion proteins. Pulled down reactions of ICP27, ORF57 fusion proteins, and GST alone with HeLa lysates were blotted with anti-GAPDH Ab, and this showed GAPDH present only in the input and not pulled down in bound samples. C , Coomassie-stained 10% SDS-PAGE of bacterially expressed proteins GST-ICP27, GST-ORF57, and GST alone present on the beads used in the binding reactions here and in Fig. 5 A . The single asterisk indicates the molecular weight of the expected protein product, whereas the double asterisks indicate cleavage products of the full-length protein.
Figure Legend Snippet: Nup62 interacts with ORF57, an ICP27 homologue from KSHV. A , mock-infected HeLa extracts were incubated with either GST-ICP27, GST-ORF57 (a homologue of ICP27 from KSHV), or GST alone. Complexes isolated on glutathione-Sepharose beads were eluted after washing, separated on 10% SDS-PAGE, transferred to nitrocellulose membranes, and incubated with Abs against Nup62. Both ICP27 homologs pulled down Nup62. 33% of the HeLa cell extracts used for binding were loaded as input, and one-half of the pull-down reaction samples eluted from the beads were loaded on the gel. The lower band marked with an asterisk in lane 2 is a band from the GST-ICP27 protein preparation cross-reacting nonspecifically with Nup62 Ab. B , control protein GAPDH did not bind to ICP27 and ORF57 fusion proteins. Pulled down reactions of ICP27, ORF57 fusion proteins, and GST alone with HeLa lysates were blotted with anti-GAPDH Ab, and this showed GAPDH present only in the input and not pulled down in bound samples. C , Coomassie-stained 10% SDS-PAGE of bacterially expressed proteins GST-ICP27, GST-ORF57, and GST alone present on the beads used in the binding reactions here and in Fig. 5 A . The single asterisk indicates the molecular weight of the expected protein product, whereas the double asterisks indicate cleavage products of the full-length protein.

Techniques Used: Infection, Incubation, Isolation, SDS Page, Binding Assay, Staining, Molecular Weight

17) Product Images from "Herpes Simplex Virus ICP27 Protein Directly Interacts with the Nuclear Pore Complex through Nup62, Inhibiting Host Nucleocytoplasmic Transport Pathways *"

Article Title: Herpes Simplex Virus ICP27 Protein Directly Interacts with the Nuclear Pore Complex through Nup62, Inhibiting Host Nucleocytoplasmic Transport Pathways *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.331777

Interaction of GST-Nup62 and ICP27 does not depend on RNA or virus infection. A , Nup62 fused to GST and purified from bacteria in the absence of any other mammalian proteins. Nup62-GST was incubated with WT HSV-1-, Δ27-, and mock-infected cell extracts or with extracts from uninfected cells expressing ICP27 from a plasmid behind the CMV promoter. Upper panel , Western blotting with Abs against ICP27. Lower panel , Western blotting with GAPDH Abs as a loading control for lysates. Input and pull-downs with GST alone were added on the ends for controls. B , GST-Nup62 was incubated with WT HSV-1-infected lysates either with or without RNase I treatment. Again, GST alone and input were used as controls. C , Rae1, another transport receptor, does not pull down ICP27. Bacterially purified GST-Rae1 (67 kDa) was incubated with lysates from WT HSV-1-, Δ27 null virus-, a mutant HSV-1 strain (M15) carrying a C-terminal point mutant (P465L/G466E) in ICP27-, or mock-infected cells. Upper panel , proteins co-purifying on glutathione-Sepharose beads were analyzed by Western blotting with Ab against ICP27. GST-Rae1 did not pull down ICP27 from any of the added cell extracts. A doublet band running lower in lanes 2–5 and marked with an asterisk is a band from the GST-Rae1 bacterial protein preparation cross-reacting nonspecifically with anti-ICP27 Ab. D , bacterially expressed GST-Rae1 protein from the fusion protein preparation used in binding assays shown in Fig. 4 C was analyzed on a Coomassie-stained gel. The expected full-length protein position is shown with an asterisk , whereas the double asterisks indicate cleavage products of the full-length protein. E , GST-Rae1 and GST-TAP pull-down known partner protein Nup98. Bacterially purified fusion protein GST-Rae1 (67 kDa) was incubated in the presence of RNase I with HSV-1 Δ27 null virus-infected or mock-infected cell lysates. Another fusion protein, GST-TAP, earlier shown to interact with Nup98, and GST alone were also incubated with HSV-1 WT virus-infected cell lysates. Proteins co-purifying on glutathione-Sepharose beads shown as bound samples and unbound leftover samples were analyzed by Western blotting with Ab against Nup98. Mock-infected and Δ27 null virus-infected cell lysates were loaded as input. Bound samples show that GST-Rae1 pulled down Nup98 from Δ27 null virus-infected or mock-infected cell lysates, and GST-TAP also pulled down Nup98 from WT virus-infected lysates. GST alone showed a weak band as background in bound samples; however, the majority of Nup98 is not pulled down and is present in unbound samples with GST alone, whereas not much is left as unbound with mock.
Figure Legend Snippet: Interaction of GST-Nup62 and ICP27 does not depend on RNA or virus infection. A , Nup62 fused to GST and purified from bacteria in the absence of any other mammalian proteins. Nup62-GST was incubated with WT HSV-1-, Δ27-, and mock-infected cell extracts or with extracts from uninfected cells expressing ICP27 from a plasmid behind the CMV promoter. Upper panel , Western blotting with Abs against ICP27. Lower panel , Western blotting with GAPDH Abs as a loading control for lysates. Input and pull-downs with GST alone were added on the ends for controls. B , GST-Nup62 was incubated with WT HSV-1-infected lysates either with or without RNase I treatment. Again, GST alone and input were used as controls. C , Rae1, another transport receptor, does not pull down ICP27. Bacterially purified GST-Rae1 (67 kDa) was incubated with lysates from WT HSV-1-, Δ27 null virus-, a mutant HSV-1 strain (M15) carrying a C-terminal point mutant (P465L/G466E) in ICP27-, or mock-infected cells. Upper panel , proteins co-purifying on glutathione-Sepharose beads were analyzed by Western blotting with Ab against ICP27. GST-Rae1 did not pull down ICP27 from any of the added cell extracts. A doublet band running lower in lanes 2–5 and marked with an asterisk is a band from the GST-Rae1 bacterial protein preparation cross-reacting nonspecifically with anti-ICP27 Ab. D , bacterially expressed GST-Rae1 protein from the fusion protein preparation used in binding assays shown in Fig. 4 C was analyzed on a Coomassie-stained gel. The expected full-length protein position is shown with an asterisk , whereas the double asterisks indicate cleavage products of the full-length protein. E , GST-Rae1 and GST-TAP pull-down known partner protein Nup98. Bacterially purified fusion protein GST-Rae1 (67 kDa) was incubated in the presence of RNase I with HSV-1 Δ27 null virus-infected or mock-infected cell lysates. Another fusion protein, GST-TAP, earlier shown to interact with Nup98, and GST alone were also incubated with HSV-1 WT virus-infected cell lysates. Proteins co-purifying on glutathione-Sepharose beads shown as bound samples and unbound leftover samples were analyzed by Western blotting with Ab against Nup98. Mock-infected and Δ27 null virus-infected cell lysates were loaded as input. Bound samples show that GST-Rae1 pulled down Nup98 from Δ27 null virus-infected or mock-infected cell lysates, and GST-TAP also pulled down Nup98 from WT virus-infected lysates. GST alone showed a weak band as background in bound samples; however, the majority of Nup98 is not pulled down and is present in unbound samples with GST alone, whereas not much is left as unbound with mock.

Techniques Used: Infection, Purification, Incubation, Expressing, Plasmid Preparation, Western Blot, Mutagenesis, Binding Assay, Staining

The Nup62-ICP27 interaction is direct, and TAP also binds ICP27 under similar conditions. A , GST-TAP pulls down ICP27 from WT HSV-1-infected cell extracts but not from extracts of cells infected with a viral ICP27 C-terminal point mutant (M15; upper panel ), whereas in the same binding reactions, GST-TAP pulls down Nup62 from WT HSV-1-infected cell extracts and from extracts of cells infected with a viral ICP27 C-terminal point mutant (M15) or with ICP27 null mutant Δ27 virus and from mock-infected cell extracts ( upper panel ). Bacterially expressed GST or GST-TAP was incubated with extracts from WT-, Δ27-, or M15-infected HSV-1 strains. Proteins co-purifying on glutathione-Sepharose beads were analyzed by Western blot with ICP27 or Nup62 Abs. B , ICP27 and TAP co-immunoprecipitate from HeLa cell extracts in the presence of RNase I. Co-immunoprecipitation was carried out by mixing anti-ICP27 antibodies 1113 and 1119 with HSV-1 WT and ICP27 mutant M15 and null Δ27 viruses and mock-infected HeLa cell extracts. Complexes formed were separated by SDS-PAGE followed by Western blotting with anti-TAP and ICP27 Abs. ICP27 co-precipitated TAP. Upper panel , co-immunoprecipitation of TAP in WT-infected HeLa cell extracts in the presence of RNase I by ICP27 but no co-immunoprecipitation with M15-, Δ27-, and mock-infected cells. Input , mock-infected HeLa cell extract. The lower band in the input lane could be due to another cellular protein cross-reacting with this TAP Ab, but the correct size upper band specific to TAP is clearly seen. The band marked with an asterisk is the heavy chain of IgGs used for immunoprecipitations. ICP27 immunoprecipitated itself. The lower panel shows immunoprecipitation of ICP27 in WT- and M15-infected HeLa cell extracts in the presence of RNase I by its Ab but no immunoprecipitation in Δ27- and mock-infected cell extracts. C , the purity of various fusion proteins used for in vitro binding assays. Coomassie-stained 10% SDS-PAGE of bacterially expressed proteins used in Figs. 4 – 7 . The single asterisk indicates the molecular weight of the expected protein product, whereas the double asterisks indicate cleavage products of the full-length protein. Prestained molecular weight protein markers are loaded on the leftmost lane with GST alone. D , bacterially purified His-tagged ICP27 was incubated with bacterially purified GST-Nup62, GST, or GST-TAP proteins on glutathione-Sepharose beads. Protein complexes formed were eluted by reduced glutathione and separated by SDS-PAGE and Western blotted with anti-His Ab. His-ICP27 interacted with GST-Nup62 and GST-TAP but not GST alone. E , an unrelated nonspecific His-tagged fusion protein (His-mRFP) does not interact with GST-tagged TAP or Nup62 fusion proteins. Bacterially purified His-tagged mRFP fusion protein was incubated with bacterially purified GST-TAP, GST-Nup62, or GST alone on glutathione-Sepharose beads in the presence of RNase I. Protein complexes eluted by reduced glutathione and separated by SDS-PAGE are shown on a Coomassie-stained gel. His-mRFP (corresponding to the band in input) does not interact with GST-TAP, GST-Nup62, and GST alone in bound samples run on gel ( upper panel ), but His-mRFP is present in all unbound pull-down samples run on Coomassie ( lower panel ). Purified His-mRFP alone protein added to the pull-down reactions was loaded as input. Molecular weight protein marker sizes are given in kDa on the left .
Figure Legend Snippet: The Nup62-ICP27 interaction is direct, and TAP also binds ICP27 under similar conditions. A , GST-TAP pulls down ICP27 from WT HSV-1-infected cell extracts but not from extracts of cells infected with a viral ICP27 C-terminal point mutant (M15; upper panel ), whereas in the same binding reactions, GST-TAP pulls down Nup62 from WT HSV-1-infected cell extracts and from extracts of cells infected with a viral ICP27 C-terminal point mutant (M15) or with ICP27 null mutant Δ27 virus and from mock-infected cell extracts ( upper panel ). Bacterially expressed GST or GST-TAP was incubated with extracts from WT-, Δ27-, or M15-infected HSV-1 strains. Proteins co-purifying on glutathione-Sepharose beads were analyzed by Western blot with ICP27 or Nup62 Abs. B , ICP27 and TAP co-immunoprecipitate from HeLa cell extracts in the presence of RNase I. Co-immunoprecipitation was carried out by mixing anti-ICP27 antibodies 1113 and 1119 with HSV-1 WT and ICP27 mutant M15 and null Δ27 viruses and mock-infected HeLa cell extracts. Complexes formed were separated by SDS-PAGE followed by Western blotting with anti-TAP and ICP27 Abs. ICP27 co-precipitated TAP. Upper panel , co-immunoprecipitation of TAP in WT-infected HeLa cell extracts in the presence of RNase I by ICP27 but no co-immunoprecipitation with M15-, Δ27-, and mock-infected cells. Input , mock-infected HeLa cell extract. The lower band in the input lane could be due to another cellular protein cross-reacting with this TAP Ab, but the correct size upper band specific to TAP is clearly seen. The band marked with an asterisk is the heavy chain of IgGs used for immunoprecipitations. ICP27 immunoprecipitated itself. The lower panel shows immunoprecipitation of ICP27 in WT- and M15-infected HeLa cell extracts in the presence of RNase I by its Ab but no immunoprecipitation in Δ27- and mock-infected cell extracts. C , the purity of various fusion proteins used for in vitro binding assays. Coomassie-stained 10% SDS-PAGE of bacterially expressed proteins used in Figs. 4 – 7 . The single asterisk indicates the molecular weight of the expected protein product, whereas the double asterisks indicate cleavage products of the full-length protein. Prestained molecular weight protein markers are loaded on the leftmost lane with GST alone. D , bacterially purified His-tagged ICP27 was incubated with bacterially purified GST-Nup62, GST, or GST-TAP proteins on glutathione-Sepharose beads. Protein complexes formed were eluted by reduced glutathione and separated by SDS-PAGE and Western blotted with anti-His Ab. His-ICP27 interacted with GST-Nup62 and GST-TAP but not GST alone. E , an unrelated nonspecific His-tagged fusion protein (His-mRFP) does not interact with GST-tagged TAP or Nup62 fusion proteins. Bacterially purified His-tagged mRFP fusion protein was incubated with bacterially purified GST-TAP, GST-Nup62, or GST alone on glutathione-Sepharose beads in the presence of RNase I. Protein complexes eluted by reduced glutathione and separated by SDS-PAGE are shown on a Coomassie-stained gel. His-mRFP (corresponding to the band in input) does not interact with GST-TAP, GST-Nup62, and GST alone in bound samples run on gel ( upper panel ), but His-mRFP is present in all unbound pull-down samples run on Coomassie ( lower panel ). Purified His-mRFP alone protein added to the pull-down reactions was loaded as input. Molecular weight protein marker sizes are given in kDa on the left .

Techniques Used: Infection, Mutagenesis, Binding Assay, Incubation, Western Blot, Immunoprecipitation, SDS Page, In Vitro, Staining, Molecular Weight, Purification, Marker

Mapping of Nup62 interaction sites on ICP27. A, schematic of ICP27, N-terminal deletion mutants, and C-terminal point mutants. The NES, NLS, and the RGG viral RNA binding site are marked along with the three hnRNP K homology domains ( KH1 to - 3 ). In the third conserved domain are the Sm and zinc finger regions that are disrupted by the M15 (P465L/G466E) and M16 point mutations (C488L) ( 57 ), respectively, and are represented with double and single asterisks , respectively. B , GST-Nup62 was incubated with lysates from cells infected with herpesviruses carrying the listed ICP27 mutations. The material that bound to GST-Nup62 and eluted from glutathione-Sepharose beads is shown in the upper panels , whereas the unbound material is shown in the lower panels to confirm that the viral proteins were expressed. All co-precipitated proteins are visualized with the ICP27 monoclonal Abs, either 1113 or 1119 because 1113 Ab is raised against a region of ICP27 that is missing in mutant d3–4 and 1119 Ab is raised against a region that is missing in dleu mutant. Standard GST alone and input controls were employed.
Figure Legend Snippet: Mapping of Nup62 interaction sites on ICP27. A, schematic of ICP27, N-terminal deletion mutants, and C-terminal point mutants. The NES, NLS, and the RGG viral RNA binding site are marked along with the three hnRNP K homology domains ( KH1 to - 3 ). In the third conserved domain are the Sm and zinc finger regions that are disrupted by the M15 (P465L/G466E) and M16 point mutations (C488L) ( 57 ), respectively, and are represented with double and single asterisks , respectively. B , GST-Nup62 was incubated with lysates from cells infected with herpesviruses carrying the listed ICP27 mutations. The material that bound to GST-Nup62 and eluted from glutathione-Sepharose beads is shown in the upper panels , whereas the unbound material is shown in the lower panels to confirm that the viral proteins were expressed. All co-precipitated proteins are visualized with the ICP27 monoclonal Abs, either 1113 or 1119 because 1113 Ab is raised against a region of ICP27 that is missing in mutant d3–4 and 1119 Ab is raised against a region that is missing in dleu mutant. Standard GST alone and input controls were employed.

Techniques Used: RNA Binding Assay, Incubation, Infection, Mutagenesis

Nup62 interacts with ORF57, an ICP27 homologue from KSHV. A , mock-infected HeLa extracts were incubated with either GST-ICP27, GST-ORF57 (a homologue of ICP27 from KSHV), or GST alone. Complexes isolated on glutathione-Sepharose beads were eluted after washing, separated on 10% SDS-PAGE, transferred to nitrocellulose membranes, and incubated with Abs against Nup62. Both ICP27 homologs pulled down Nup62. 33% of the HeLa cell extracts used for binding were loaded as input, and one-half of the pull-down reaction samples eluted from the beads were loaded on the gel. The lower band marked with an asterisk in lane 2 is a band from the GST-ICP27 protein preparation cross-reacting nonspecifically with Nup62 Ab. B , control protein GAPDH did not bind to ICP27 and ORF57 fusion proteins. Pulled down reactions of ICP27, ORF57 fusion proteins, and GST alone with HeLa lysates were blotted with anti-GAPDH Ab, and this showed GAPDH present only in the input and not pulled down in bound samples. C , Coomassie-stained 10% SDS-PAGE of bacterially expressed proteins GST-ICP27, GST-ORF57, and GST alone present on the beads used in the binding reactions here and in Fig. 5 A . The single asterisk indicates the molecular weight of the expected protein product, whereas the double asterisks indicate cleavage products of the full-length protein.
Figure Legend Snippet: Nup62 interacts with ORF57, an ICP27 homologue from KSHV. A , mock-infected HeLa extracts were incubated with either GST-ICP27, GST-ORF57 (a homologue of ICP27 from KSHV), or GST alone. Complexes isolated on glutathione-Sepharose beads were eluted after washing, separated on 10% SDS-PAGE, transferred to nitrocellulose membranes, and incubated with Abs against Nup62. Both ICP27 homologs pulled down Nup62. 33% of the HeLa cell extracts used for binding were loaded as input, and one-half of the pull-down reaction samples eluted from the beads were loaded on the gel. The lower band marked with an asterisk in lane 2 is a band from the GST-ICP27 protein preparation cross-reacting nonspecifically with Nup62 Ab. B , control protein GAPDH did not bind to ICP27 and ORF57 fusion proteins. Pulled down reactions of ICP27, ORF57 fusion proteins, and GST alone with HeLa lysates were blotted with anti-GAPDH Ab, and this showed GAPDH present only in the input and not pulled down in bound samples. C , Coomassie-stained 10% SDS-PAGE of bacterially expressed proteins GST-ICP27, GST-ORF57, and GST alone present on the beads used in the binding reactions here and in Fig. 5 A . The single asterisk indicates the molecular weight of the expected protein product, whereas the double asterisks indicate cleavage products of the full-length protein.

Techniques Used: Infection, Incubation, Isolation, SDS Page, Binding Assay, Staining, Molecular Weight

18) Product Images from "Ponsin/SH3P12: An l-Afadin- and Vinculin-binding Protein Localized at Cell-Cell and Cell-Matrix Adherens Junctions "

Article Title: Ponsin/SH3P12: An l-Afadin- and Vinculin-binding Protein Localized at Cell-Cell and Cell-Matrix Adherens Junctions

Journal: The Journal of Cell Biology

doi:

Inability of ponsin to form a ternary complex with l-afadin and vinculin. (A) Immunoprecipitation from COS7 cells. Myc– l-afadin (the full length), FLAG–ponsin-2 (full length), and Myc–vinculin-C (the COOH-terminal half) were expressed in COS7 cells in various combinations by transfection with pCMV-Myc–l-afadin, pFLAG-CMV2–ponsin-2, and pCMV-Myc–vinculin-C, respectively. Each cell extract was subjected to immunoprecipitation with the anti–Myc or anti–FLAG antibody. The precipitate was then subjected to SDS-PAGE (8% polyacrylamide gel), followed by Western blot analysis using the anti–Myc, anti–FLAG, anti–l-afadin, or anti-vinculin antibody. (A1) COS7 cells expressing FLAG–ponsin-2 alone, (A2) COS7 cells expressing both FLAG–ponsin-2 and Myc–vinculin-C, (A3) COS7 cells expressing both Myc–l-afadin and FLAG–ponsin-2. (IP) Immunoprecipitation. (B) Affinity chromatography using ponsin-immobilized beads. Various doses of His6–l-afadin-C199 (the COOH-terminal 199 aa region including the third proline-rich region) and MBP–vinculin-C (the COOH-terminal half) were mixed and incubated with GST–ponsin-2-C (the COOH-terminal half) immobilized on glutathione-Sepharose beads. Each eluate was subjected to SDS-PAGE (15% polyacrylamide gel), followed by protein staining with Coomassie brilliant blue. The results are representative of three independent experiments.
Figure Legend Snippet: Inability of ponsin to form a ternary complex with l-afadin and vinculin. (A) Immunoprecipitation from COS7 cells. Myc– l-afadin (the full length), FLAG–ponsin-2 (full length), and Myc–vinculin-C (the COOH-terminal half) were expressed in COS7 cells in various combinations by transfection with pCMV-Myc–l-afadin, pFLAG-CMV2–ponsin-2, and pCMV-Myc–vinculin-C, respectively. Each cell extract was subjected to immunoprecipitation with the anti–Myc or anti–FLAG antibody. The precipitate was then subjected to SDS-PAGE (8% polyacrylamide gel), followed by Western blot analysis using the anti–Myc, anti–FLAG, anti–l-afadin, or anti-vinculin antibody. (A1) COS7 cells expressing FLAG–ponsin-2 alone, (A2) COS7 cells expressing both FLAG–ponsin-2 and Myc–vinculin-C, (A3) COS7 cells expressing both Myc–l-afadin and FLAG–ponsin-2. (IP) Immunoprecipitation. (B) Affinity chromatography using ponsin-immobilized beads. Various doses of His6–l-afadin-C199 (the COOH-terminal 199 aa region including the third proline-rich region) and MBP–vinculin-C (the COOH-terminal half) were mixed and incubated with GST–ponsin-2-C (the COOH-terminal half) immobilized on glutathione-Sepharose beads. Each eluate was subjected to SDS-PAGE (15% polyacrylamide gel), followed by protein staining with Coomassie brilliant blue. The results are representative of three independent experiments.

Techniques Used: Immunoprecipitation, Transfection, SDS Page, Western Blot, Expressing, Affinity Chromatography, Incubation, Staining

Binding regions of ponsin and vinculin. (A) Blot overlay. (A1) 35 S-Labeled vinculin blot overlay. Various GST-fused proteins of ponsin-2 (5 pmol each) were subjected to SDS-PAGE (8% polyacrylamide gel), followed by 35 S-labeled vinculin blot overlay. (A2) 35 S- Labeled ponsin-2 blot overlay. Full-length native vinculin and various recombinant proteins of vinculin (5 pmol each) were subjected to SDS-PAGE (8 and 12% discontinuous polyacrylamide gel), followed by 35 S- labeled ponsin blot overlay. (B) Affinity chromatography. (B1) Binding region of ponsin. MBP– vinculin-C (the COOH-terminal half) was incubated with various GST-fused proteins of ponsin-2 immobilized on glutathione-Sepharose beads. Each eluate was subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blot analysis using the anti-vinculin antibody. (B2) Binding region of vinculin. MBP–vinculin-C or MBP alone was incubated with GST–ponsin-2-C (the COOH-terminal half) or GST alone immobilized on glutathione-Sepharose beads. Each original sample and eluate were subjected to SDS-PAGE (12% polyacrylamide gel), followed by protein staining with Coomassie brilliant blue. (F) GST– ponsin-2-F (the full length), (N) GST–ponsin-2-N (the NH 2 -terminal half), (C) GST–ponsin-2-C, [SH3(1+2)] GST–ponsin-2-SH3(1+2) (the first and second SH3 domains), [SH3(2+3)] GST–ponsin-2-SH3(2+3) (the second and third SH3 domains), [SH3(1)] GST–ponsin-2-SH3(1) (the first SH3 domain), [SH3(2)] GST–ponsin-2-SH3(2) (the second SH3 domain), [SH3(3)] GST–ponsin-2-SH3(3) (the third SH3 domain), [PR] the proline-rich region of vinculin (aa 837–878), (1) Myc–vinculin-1 (the NH 2 -terminal region, aa 1–800), (2) Myc–vinculin-2 (the COOH-terminal region, aa 801–1066); (P) GST–vinculin-P (the proline-rich region), and (C-ΔP) GST–vinculin-C-ΔP (the COOH-terminal region lacking the proline-rich region). The results are representative of three independent experiments.
Figure Legend Snippet: Binding regions of ponsin and vinculin. (A) Blot overlay. (A1) 35 S-Labeled vinculin blot overlay. Various GST-fused proteins of ponsin-2 (5 pmol each) were subjected to SDS-PAGE (8% polyacrylamide gel), followed by 35 S-labeled vinculin blot overlay. (A2) 35 S- Labeled ponsin-2 blot overlay. Full-length native vinculin and various recombinant proteins of vinculin (5 pmol each) were subjected to SDS-PAGE (8 and 12% discontinuous polyacrylamide gel), followed by 35 S- labeled ponsin blot overlay. (B) Affinity chromatography. (B1) Binding region of ponsin. MBP– vinculin-C (the COOH-terminal half) was incubated with various GST-fused proteins of ponsin-2 immobilized on glutathione-Sepharose beads. Each eluate was subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blot analysis using the anti-vinculin antibody. (B2) Binding region of vinculin. MBP–vinculin-C or MBP alone was incubated with GST–ponsin-2-C (the COOH-terminal half) or GST alone immobilized on glutathione-Sepharose beads. Each original sample and eluate were subjected to SDS-PAGE (12% polyacrylamide gel), followed by protein staining with Coomassie brilliant blue. (F) GST– ponsin-2-F (the full length), (N) GST–ponsin-2-N (the NH 2 -terminal half), (C) GST–ponsin-2-C, [SH3(1+2)] GST–ponsin-2-SH3(1+2) (the first and second SH3 domains), [SH3(2+3)] GST–ponsin-2-SH3(2+3) (the second and third SH3 domains), [SH3(1)] GST–ponsin-2-SH3(1) (the first SH3 domain), [SH3(2)] GST–ponsin-2-SH3(2) (the second SH3 domain), [SH3(3)] GST–ponsin-2-SH3(3) (the third SH3 domain), [PR] the proline-rich region of vinculin (aa 837–878), (1) Myc–vinculin-1 (the NH 2 -terminal region, aa 1–800), (2) Myc–vinculin-2 (the COOH-terminal region, aa 801–1066); (P) GST–vinculin-P (the proline-rich region), and (C-ΔP) GST–vinculin-C-ΔP (the COOH-terminal region lacking the proline-rich region). The results are representative of three independent experiments.

Techniques Used: Binding Assay, Labeling, SDS Page, Recombinant, Affinity Chromatography, Incubation, Western Blot, Staining

Binding of l-afadin to ponsin in vivo. (A) Immunoprecipitation analysis of l-afadin and ponsin-2. FLAG–ponsin-2 (the full length) and Myc–l-afadin (the full length) were coexpressed in COS7 cells in various combinations by transfection with pFLAG-CMV2–ponsin-2 and pCMV-Myc–l-afadin, respectively. Each cell extract was subjected to immunoprecipitation with the anti–FLAG or anti–Myc antibody. The precipitate was then subjected to SDS-PAGE (8% polyacrylamide gel), followed by Western blot analysis using the anti–FLAG or anti–Myc antibody, or by protein staining with Coomassie brilliant blue. (A1) Western blot analysis, and (A2) protein staining. (IP) Immunoprecipitation, and (CBB) staining with Coomassie brilliant blue. (B) Immunoprecipitation analysis of s-afadin and ponsin-2. Both FLAG–ponsin-2 and tag-free l-afadin (the full length) or both FLAG–ponsin-2 and tag-free s-afadin (the full length) were coexpressed in COS7 cells by transfection with both pFLAG-CMV2– ponsin-2 and pCMV5–l-afadin or both pFLAG-CMV2–ponsin-2 and pCMV5–s-afadin, respectively. Each cell extract was incubated with the anti–FLAG antibody and protein G–Sepharose beads, followed by centrifugation. The beads and the supernatant were separately subjected to SDS-PAGE (8% polyacrylamide gel), followed by Western blot analysis using the anti–l- and s-afadin antibody. (Sup) supernatant. The results are representative of three independent experiments.
Figure Legend Snippet: Binding of l-afadin to ponsin in vivo. (A) Immunoprecipitation analysis of l-afadin and ponsin-2. FLAG–ponsin-2 (the full length) and Myc–l-afadin (the full length) were coexpressed in COS7 cells in various combinations by transfection with pFLAG-CMV2–ponsin-2 and pCMV-Myc–l-afadin, respectively. Each cell extract was subjected to immunoprecipitation with the anti–FLAG or anti–Myc antibody. The precipitate was then subjected to SDS-PAGE (8% polyacrylamide gel), followed by Western blot analysis using the anti–FLAG or anti–Myc antibody, or by protein staining with Coomassie brilliant blue. (A1) Western blot analysis, and (A2) protein staining. (IP) Immunoprecipitation, and (CBB) staining with Coomassie brilliant blue. (B) Immunoprecipitation analysis of s-afadin and ponsin-2. Both FLAG–ponsin-2 and tag-free l-afadin (the full length) or both FLAG–ponsin-2 and tag-free s-afadin (the full length) were coexpressed in COS7 cells by transfection with both pFLAG-CMV2– ponsin-2 and pCMV5–l-afadin or both pFLAG-CMV2–ponsin-2 and pCMV5–s-afadin, respectively. Each cell extract was incubated with the anti–FLAG antibody and protein G–Sepharose beads, followed by centrifugation. The beads and the supernatant were separately subjected to SDS-PAGE (8% polyacrylamide gel), followed by Western blot analysis using the anti–l- and s-afadin antibody. (Sup) supernatant. The results are representative of three independent experiments.

Techniques Used: Binding Assay, In Vivo, Immunoprecipitation, Transfection, SDS Page, Western Blot, Staining, Incubation, Centrifugation

Binding regions of l-afadin and ponsin. (A) Blot overlay. (A1) 35 S-Labeled ponsin-2 blot overlay. Myc–l-Afadin (the full length), Myc–s-afadin (the full length), His6–l-afadin-C199 (the COOH-terminal 199 aa region including the third proline-rich region), and His6– l-afadin-C132 (the COOH-terminal 132 aa region lacking the aa residues [PPLP] of the third proline-rich domain) (5 pmol each) were subjected to SDS-PAGE (8 and 12% discontinuous polyacrylamide gel), followed by 35 S-labeled ponsin-2 blot overlay. (A2) 35 S-Labeled l-afadin blot overlay. Various GST-fused proteins of ponsin-2 (5 pmol each) were subjected to SDS-PAGE (8% polyacrylamide gel), followed by 35 S-labeled l-afadin blot overlay. (B) Affinity chromatography. (B1) Binding region of l-afadin. His6–l-Afadin-C199 or His6–l-afadin-C132 was incubated with GST–ponsin-2-C (the COOH-terminal half) or GST alone immobilized on glutathione-Sepharose beads. Each original sample and eluate were subjected to SDS-PAGE (15% polyacrylamide gel), followed by protein staining with Coomassie brilliant blue. (B2) Binding region of ponsin. His6– l-afadin-C199 was incubated with various GST-fused proteins of ponsin-2 immobilized on glutathione-Sepharose beads. Each eluate was subjected to SDS-PAGE (12% polyacrylamide gel), followed by Western blot analysis using the anti–l-afadin antibody. [PR(1)] the first proline-rich region (aa 1219–1229), [PR(2)] the second proline-rich region (aa 1372–1399), [PR(3)] the third proline-rich region (aa 1691–1713), (C199) His6–l-afadin-C199, (C132) His6–l-afadin-C132, (F) GST–ponsin-2-F (the full length), (N) GST–ponsin-2-N (the NH 2 -terminal half), (C) GST–ponsin-2-C, [SH3(1+2)] GST– ponsin-2-SH3(1+2) (the first and second SH3 domains), [SH3(2+3)] GST–ponsin-2-SH3(2+3) (the second and third SH3 domains), [SH3(1)] GST–ponsin-2-SH3(1) (the first SH3 domain), [SH3(2)] GST–ponsin-2-SH3(2) (the second SH3 domain), and [SH3(3)] GST– ponsin-2-SH3(3) (the third SH3 domain). The results are representative of three independent experiments.
Figure Legend Snippet: Binding regions of l-afadin and ponsin. (A) Blot overlay. (A1) 35 S-Labeled ponsin-2 blot overlay. Myc–l-Afadin (the full length), Myc–s-afadin (the full length), His6–l-afadin-C199 (the COOH-terminal 199 aa region including the third proline-rich region), and His6– l-afadin-C132 (the COOH-terminal 132 aa region lacking the aa residues [PPLP] of the third proline-rich domain) (5 pmol each) were subjected to SDS-PAGE (8 and 12% discontinuous polyacrylamide gel), followed by 35 S-labeled ponsin-2 blot overlay. (A2) 35 S-Labeled l-afadin blot overlay. Various GST-fused proteins of ponsin-2 (5 pmol each) were subjected to SDS-PAGE (8% polyacrylamide gel), followed by 35 S-labeled l-afadin blot overlay. (B) Affinity chromatography. (B1) Binding region of l-afadin. His6–l-Afadin-C199 or His6–l-afadin-C132 was incubated with GST–ponsin-2-C (the COOH-terminal half) or GST alone immobilized on glutathione-Sepharose beads. Each original sample and eluate were subjected to SDS-PAGE (15% polyacrylamide gel), followed by protein staining with Coomassie brilliant blue. (B2) Binding region of ponsin. His6– l-afadin-C199 was incubated with various GST-fused proteins of ponsin-2 immobilized on glutathione-Sepharose beads. Each eluate was subjected to SDS-PAGE (12% polyacrylamide gel), followed by Western blot analysis using the anti–l-afadin antibody. [PR(1)] the first proline-rich region (aa 1219–1229), [PR(2)] the second proline-rich region (aa 1372–1399), [PR(3)] the third proline-rich region (aa 1691–1713), (C199) His6–l-afadin-C199, (C132) His6–l-afadin-C132, (F) GST–ponsin-2-F (the full length), (N) GST–ponsin-2-N (the NH 2 -terminal half), (C) GST–ponsin-2-C, [SH3(1+2)] GST– ponsin-2-SH3(1+2) (the first and second SH3 domains), [SH3(2+3)] GST–ponsin-2-SH3(2+3) (the second and third SH3 domains), [SH3(1)] GST–ponsin-2-SH3(1) (the first SH3 domain), [SH3(2)] GST–ponsin-2-SH3(2) (the second SH3 domain), and [SH3(3)] GST– ponsin-2-SH3(3) (the third SH3 domain). The results are representative of three independent experiments.

Techniques Used: Binding Assay, Labeling, SDS Page, Affinity Chromatography, Incubation, Staining, Western Blot

19) Product Images from "DnaJA1/Hsp40 Is Co-Opted by Influenza A Virus To Enhance Its Viral RNA Polymerase Activity"

Article Title: DnaJA1/Hsp40 Is Co-Opted by Influenza A Virus To Enhance Its Viral RNA Polymerase Activity

Journal: Journal of Virology

doi: 10.1128/JVI.02475-14

DnaJA1 associates with PB2 and PA subunits. (A) Flag-tagged DnaJA1 copurifies with both PB2 and PA. The control vector, TAP-tagged WSN PA, PB1, PB2, or NP expression plasmids were individually transfected into 293T cells along with Flag-tagged DnaJA1. At 40 h posttransfection, cells were harvested and lysed, followed by IgG Sepharose purification and Western blot analyses. IP, immunoprecipitation. (B) Endogenous DnaJA1 interacts with PB2 and PA. The control vector, TAP-tagged WSN PA, PB1, PB2, and NP expression constructs were individually transfected into 293T cells. At 40 h posttransfection, cells were harvested and lysed, followed by IgG Sepharose purification and Western blot analyses with an anti-DnaJA1 antibody. (C) Endogenous DnaJA1 interacts with PB2 and PA in virus-infected cells. 293T cells were infected with influenza WSN virus (MO1 = 0.5). Cells were lysed at 16 h.p.i. and subjected to immunoprecipitation with either normal IgG or anti-PB2/anti-PA antibodies, followed by Western blot analyses with an anti-DnaJA1 antibody. (D) Exogenous DnaJA1 interacts with PB2 and PA. 293T cells were transfected with the pcDNA-PB1, pcDNA-PB2. and pcDNA-PA for 40 h. Cells were lysed and subjected to GST pulldown assay with the recombinant GST or GST-DnaJA1 prepared from E. coli cells. The pulldown samples were analyzed by Western blotting with anti-PB2 or anti-PA antibody. Coomassie brilliant blue (CBB) staining of purified protein is shown at the bottom.
Figure Legend Snippet: DnaJA1 associates with PB2 and PA subunits. (A) Flag-tagged DnaJA1 copurifies with both PB2 and PA. The control vector, TAP-tagged WSN PA, PB1, PB2, or NP expression plasmids were individually transfected into 293T cells along with Flag-tagged DnaJA1. At 40 h posttransfection, cells were harvested and lysed, followed by IgG Sepharose purification and Western blot analyses. IP, immunoprecipitation. (B) Endogenous DnaJA1 interacts with PB2 and PA. The control vector, TAP-tagged WSN PA, PB1, PB2, and NP expression constructs were individually transfected into 293T cells. At 40 h posttransfection, cells were harvested and lysed, followed by IgG Sepharose purification and Western blot analyses with an anti-DnaJA1 antibody. (C) Endogenous DnaJA1 interacts with PB2 and PA in virus-infected cells. 293T cells were infected with influenza WSN virus (MO1 = 0.5). Cells were lysed at 16 h.p.i. and subjected to immunoprecipitation with either normal IgG or anti-PB2/anti-PA antibodies, followed by Western blot analyses with an anti-DnaJA1 antibody. (D) Exogenous DnaJA1 interacts with PB2 and PA. 293T cells were transfected with the pcDNA-PB1, pcDNA-PB2. and pcDNA-PA for 40 h. Cells were lysed and subjected to GST pulldown assay with the recombinant GST or GST-DnaJA1 prepared from E. coli cells. The pulldown samples were analyzed by Western blotting with anti-PB2 or anti-PA antibody. Coomassie brilliant blue (CBB) staining of purified protein is shown at the bottom.

Techniques Used: Plasmid Preparation, Expressing, Transfection, Purification, Western Blot, Immunoprecipitation, Construct, Infection, GST Pulldown Assay, Recombinant, Staining

Identification of DnaJA1 as a positive regulator for influenza virus replication. (A) DnaJA1 copurifies with the PB2 subunit. 293T cells were transfected with pcDNA-PB2-TAP or pcDNA-TAP-NP. At 40 h posttransfection, cells were lysed and subjected to IgG Sepharose purification. The purified proteins were released with TEV protease. A small of amount of the purified proteins were resolved on an 8% PAGE gel and visualized by silver staining. The rest were subjected to nano-LC-MS/MS analysis. There were 23 proteins which overlapped in both samples and are not listed individually. Preliminary proteins that were identified as either PB2-TAP sample-specific proteins or TAP-NP sample-specific proteins are shown in the boxes. (B) DnaJA1 acts as positive regulator specifically for influenza virus replication. A549 cells treated with DnaJA1-specific siRNA or control siRNA were infected with either influenza WSN virus or VSV at an MOI of 0.001. Every 12 h postinfection, the viral titers in the supernatant were determined by plaque assay on MDCK cells. The values are means of results from three independent experiments. Representative Western blot results for examining the knockdown efficiency of DnaJA1 are shown. (C) Knockdown of DnaJA1 reduces influenza virus protein expression. A549 cells treated with DnaJA1-specific siRNA or control siRNA were infected with influenza A/WSN/33 virus (MOI = 1). At the indicated times points postinfection, cells were harvested and lysed for Western blot analysis with NP- and M1-specific antibodies. (D) Statistical analysis of NP and M1 levels in panel C. Values for NP and M1 were standardized to the actin level and normalized to the levels of NP and M1 in cells treated with control siRNA (means ± SEMs of results from three independent experiments). (E) Overexpression of DnaJA1 increases influenza virus protein expression. 293T cells were transfected with pCMV-DnaJA1-Flag or pCMV6 empty vector for 24 h, followed by infection with WSN virus (MOI = 1). At the indicated times postinfection, cells were harvested and lysed for Western blot analysis with NP- and M1-specific antibodies. (F) Statistical analysis of NP and M1 levels in panel E. Values for NP and M1 were standardized to the actin level and normalized to levels of NP and M1 in cells transfected with control vector (means ± SEMs of the results from three independent experiments). *, P
Figure Legend Snippet: Identification of DnaJA1 as a positive regulator for influenza virus replication. (A) DnaJA1 copurifies with the PB2 subunit. 293T cells were transfected with pcDNA-PB2-TAP or pcDNA-TAP-NP. At 40 h posttransfection, cells were lysed and subjected to IgG Sepharose purification. The purified proteins were released with TEV protease. A small of amount of the purified proteins were resolved on an 8% PAGE gel and visualized by silver staining. The rest were subjected to nano-LC-MS/MS analysis. There were 23 proteins which overlapped in both samples and are not listed individually. Preliminary proteins that were identified as either PB2-TAP sample-specific proteins or TAP-NP sample-specific proteins are shown in the boxes. (B) DnaJA1 acts as positive regulator specifically for influenza virus replication. A549 cells treated with DnaJA1-specific siRNA or control siRNA were infected with either influenza WSN virus or VSV at an MOI of 0.001. Every 12 h postinfection, the viral titers in the supernatant were determined by plaque assay on MDCK cells. The values are means of results from three independent experiments. Representative Western blot results for examining the knockdown efficiency of DnaJA1 are shown. (C) Knockdown of DnaJA1 reduces influenza virus protein expression. A549 cells treated with DnaJA1-specific siRNA or control siRNA were infected with influenza A/WSN/33 virus (MOI = 1). At the indicated times points postinfection, cells were harvested and lysed for Western blot analysis with NP- and M1-specific antibodies. (D) Statistical analysis of NP and M1 levels in panel C. Values for NP and M1 were standardized to the actin level and normalized to the levels of NP and M1 in cells treated with control siRNA (means ± SEMs of results from three independent experiments). (E) Overexpression of DnaJA1 increases influenza virus protein expression. 293T cells were transfected with pCMV-DnaJA1-Flag or pCMV6 empty vector for 24 h, followed by infection with WSN virus (MOI = 1). At the indicated times postinfection, cells were harvested and lysed for Western blot analysis with NP- and M1-specific antibodies. (F) Statistical analysis of NP and M1 levels in panel E. Values for NP and M1 were standardized to the actin level and normalized to levels of NP and M1 in cells transfected with control vector (means ± SEMs of the results from three independent experiments). *, P

Techniques Used: Transfection, Purification, Polyacrylamide Gel Electrophoresis, Silver Staining, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Infection, Plaque Assay, Western Blot, Expressing, Over Expression, Plasmid Preparation

DnaJA1 is recruited into the nucleus upon influenza virus infection. (A) DnaJA1 translocates into the nucleus during influenza virus replication. A549 cells were infected with WSN virus at an MOI of 3. At the indicated times postinfection, cells were stained for nuclei (blue), PB2 (green), and DnaJA1 (red) by indirect immunofluorescence assay. (B) DnaJA1 translocates into the nucleus along with PB1-PA dimer. HeLa cells were transfected with either pcDNA-PB2 or pcDNA-PA or with a combination of pcDNA-PB1 and pcDNA-PA plasmids for 48 h. Cells were stained for nuclei (blue), PB2 or PA (green), and DnaJA1 (red). (C) DnaJA1 does not affect viral RNA polymerase assembly. 293T cells were transfected with the indicated plasmids. Cell lysates were purified with IgG Sepharose, and the lysates and products were analyzed by Western blot analysis. (D) DnaJA1 does not affect viral RNP assembly. 293T cells were transfected with the indicated plasmids. Cell lysates were purified with IgG Sepharose, and the lysates and products were analyzed by Western blot analysis.
Figure Legend Snippet: DnaJA1 is recruited into the nucleus upon influenza virus infection. (A) DnaJA1 translocates into the nucleus during influenza virus replication. A549 cells were infected with WSN virus at an MOI of 3. At the indicated times postinfection, cells were stained for nuclei (blue), PB2 (green), and DnaJA1 (red) by indirect immunofluorescence assay. (B) DnaJA1 translocates into the nucleus along with PB1-PA dimer. HeLa cells were transfected with either pcDNA-PB2 or pcDNA-PA or with a combination of pcDNA-PB1 and pcDNA-PA plasmids for 48 h. Cells were stained for nuclei (blue), PB2 or PA (green), and DnaJA1 (red). (C) DnaJA1 does not affect viral RNA polymerase assembly. 293T cells were transfected with the indicated plasmids. Cell lysates were purified with IgG Sepharose, and the lysates and products were analyzed by Western blot analysis. (D) DnaJA1 does not affect viral RNP assembly. 293T cells were transfected with the indicated plasmids. Cell lysates were purified with IgG Sepharose, and the lysates and products were analyzed by Western blot analysis.

Techniques Used: Infection, Staining, Immunofluorescence, Transfection, Purification, Western Blot

20) Product Images from "ERK signaling promotes cell motility by inducing the localization of myosin 1E to lamellipodial tips"

Article Title: ERK signaling promotes cell motility by inducing the localization of myosin 1E to lamellipodial tips

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201503123

SH3P2 interacts with Myo1E via its N-terminal proline-rich region and C-terminal acidic amino acid cluster. (A) Glutathione-Sepharose beads coupled with GST or GST-tagged wild-type or indicated mutant forms of human SH3P2 were incubated with MKN1 cell lysates, after which bead-bound proteins as well as the whole-cell lysates (10% of the input for the pull-down assays) were subjected to SDS-PAGE followed by staining with Coomassie brilliant blue (CBB) or immunoblot analysis (IB) with antibodies to Myo1E. Arrowheads indicate the Myo1E band. (B) MKN1 cells were transfected for 24 h with vectors encoding EGFP-tagged wild-type or mutant forms of human SH3P2, lysed, and subjected to immunoprecipitation (IP) with antibodies to GFP. The resulting precipitates as well as the whole-cell lysates (10% of the input for immunoprecipitation) were subjected to immunoblot analysis with antibodies to Myo1E and to GFP. Amino acid sequences of the PR region and the C-terminal acidic amino acid cluster are shown. The RSK phosphorylation site (Ser 202 ), acidic amino acids, basic amino acids, and proline are indicated in yellow, red, blue, and green, respectively. All data are representative of at least three separate experiments.
Figure Legend Snippet: SH3P2 interacts with Myo1E via its N-terminal proline-rich region and C-terminal acidic amino acid cluster. (A) Glutathione-Sepharose beads coupled with GST or GST-tagged wild-type or indicated mutant forms of human SH3P2 were incubated with MKN1 cell lysates, after which bead-bound proteins as well as the whole-cell lysates (10% of the input for the pull-down assays) were subjected to SDS-PAGE followed by staining with Coomassie brilliant blue (CBB) or immunoblot analysis (IB) with antibodies to Myo1E. Arrowheads indicate the Myo1E band. (B) MKN1 cells were transfected for 24 h with vectors encoding EGFP-tagged wild-type or mutant forms of human SH3P2, lysed, and subjected to immunoprecipitation (IP) with antibodies to GFP. The resulting precipitates as well as the whole-cell lysates (10% of the input for immunoprecipitation) were subjected to immunoblot analysis with antibodies to Myo1E and to GFP. Amino acid sequences of the PR region and the C-terminal acidic amino acid cluster are shown. The RSK phosphorylation site (Ser 202 ), acidic amino acids, basic amino acids, and proline are indicated in yellow, red, blue, and green, respectively. All data are representative of at least three separate experiments.

Techniques Used: Mutagenesis, Incubation, SDS Page, Staining, Transfection, Immunoprecipitation

Myo1E interacts with SH3P2 via its SH3 domain and the C-terminal positively charged region of its TH2 domain. (A) Glutathione-Sepharose beads coupled with GST-fused human Myo1E(SH3) were incubated with recombinant human SH3P2 or SH3P2(ΔPR), after which bead-bound proteins as well as recombinant SH3P2 or SH3P2(ΔPR) (10% of the input for the pull-down assay) were subjected to SDS-PAGE followed by staining with Coomassie brilliant blue (CBB) or immunoblot analysis (IB) with antibodies to SH3P2. (B) MKN1 cells were transfected for 24 h with vectors encoding EGFP-tagged wild-type or indicated mutant forms of human Myo1E, lysed, and subjected to immunoprecipitation (IP) with antibodies to GFP. The resulting precipitates as well as the whole-cell lysates (10% of the input for immunoprecipitation) were subjected to immunoblot analysis with antibodies to SH3P2 and to GFP. The amino acid sequence of the C-terminal positively charged region of the TH2 domain of Myo1E is shown, with acidic amino acids, basic amino acids, and proline being indicated in red, blue, and green, respectively. (C) MKN1 cells were transfected for 24 h with vectors encoding EGFP-tagged wild-type or mutant forms of human Myo1E, lysed, and subjected to pull-down assays with GST or GST-fused wild-type or mutant forms of human SH3P2. Bead-bound proteins as well as the whole-cell lysates (10% of the input for the pull-down assays) were subjected to SDS-PAGE followed by staining with Coomassie brilliant blue or by immunoblot analysis with antibodies to GFP. All data are representative of at least three separate experiments.
Figure Legend Snippet: Myo1E interacts with SH3P2 via its SH3 domain and the C-terminal positively charged region of its TH2 domain. (A) Glutathione-Sepharose beads coupled with GST-fused human Myo1E(SH3) were incubated with recombinant human SH3P2 or SH3P2(ΔPR), after which bead-bound proteins as well as recombinant SH3P2 or SH3P2(ΔPR) (10% of the input for the pull-down assay) were subjected to SDS-PAGE followed by staining with Coomassie brilliant blue (CBB) or immunoblot analysis (IB) with antibodies to SH3P2. (B) MKN1 cells were transfected for 24 h with vectors encoding EGFP-tagged wild-type or indicated mutant forms of human Myo1E, lysed, and subjected to immunoprecipitation (IP) with antibodies to GFP. The resulting precipitates as well as the whole-cell lysates (10% of the input for immunoprecipitation) were subjected to immunoblot analysis with antibodies to SH3P2 and to GFP. The amino acid sequence of the C-terminal positively charged region of the TH2 domain of Myo1E is shown, with acidic amino acids, basic amino acids, and proline being indicated in red, blue, and green, respectively. (C) MKN1 cells were transfected for 24 h with vectors encoding EGFP-tagged wild-type or mutant forms of human Myo1E, lysed, and subjected to pull-down assays with GST or GST-fused wild-type or mutant forms of human SH3P2. Bead-bound proteins as well as the whole-cell lysates (10% of the input for the pull-down assays) were subjected to SDS-PAGE followed by staining with Coomassie brilliant blue or by immunoblot analysis with antibodies to GFP. All data are representative of at least three separate experiments.

Techniques Used: Incubation, Recombinant, Pull Down Assay, SDS Page, Staining, Transfection, Mutagenesis, Immunoprecipitation, Sequencing

SH3P2 specifically interacts with Myo1E. (A) Glutathione-Sepharose beads coupled with GST or GST-SH3P2 were incubated with MKN1 cell lysates (+) or with the lysis buffer alone (−), after which bead-bound proteins were subjected to SDS-PAGE followed by staining with Coomassie brilliant blue (CBB). Bands corresponding to proteins of ∼120, ∼68, and ∼66 kD (arrows) pulled down by GST-SH3P2 were identified by MS analysis as Myo1E, Sam68, and hnRNP-K, respectively. (B) MKN1 cell lysates were subjected to immunoprecipitation (IP) with antibodies to SH3P2 or to Myo1E (or with control IgG), and the resulting precipitates as well as the whole-cell lysates (10% of the input for immunoprecipitation) were subjected to immunoblot analysis (IB) with antibodies to the indicated proteins. All data are representative of at least three separate experiments.
Figure Legend Snippet: SH3P2 specifically interacts with Myo1E. (A) Glutathione-Sepharose beads coupled with GST or GST-SH3P2 were incubated with MKN1 cell lysates (+) or with the lysis buffer alone (−), after which bead-bound proteins were subjected to SDS-PAGE followed by staining with Coomassie brilliant blue (CBB). Bands corresponding to proteins of ∼120, ∼68, and ∼66 kD (arrows) pulled down by GST-SH3P2 were identified by MS analysis as Myo1E, Sam68, and hnRNP-K, respectively. (B) MKN1 cell lysates were subjected to immunoprecipitation (IP) with antibodies to SH3P2 or to Myo1E (or with control IgG), and the resulting precipitates as well as the whole-cell lysates (10% of the input for immunoprecipitation) were subjected to immunoblot analysis (IB) with antibodies to the indicated proteins. All data are representative of at least three separate experiments.

Techniques Used: Incubation, Lysis, SDS Page, Staining, Mass Spectrometry, Immunoprecipitation

21) Product Images from "p160/SRC/NCoA coactivators form complexes via specific interaction of their PAS-B domain with the CID/AD1 domain"

Article Title: p160/SRC/NCoA coactivators form complexes via specific interaction of their PAS-B domain with the CID/AD1 domain

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkn029

Overexpression of the NCoA PAS-B domain or competition with the CID/AD1 inhibits contact of NCoA-1 to CBP or the STAT6-TAD. ( A ) 293T cells were transiently transfected with expression vectors encoding a FLAG-tagged CBP fragment (aa 2058–2130) or the empty FLAG-tag vector (pMX-FLAG) (each 1 µg), a 6× Myc-tagged CID/AD1 fragment of NCoA-1 (aa 804–1032) (0,1 µg) along with a vector containing a YFP fusion protein encoding NCoA-1 aa 213–462 or CFP as a control (each 3 µg). Whole cell extracts were prepared and co-immunoprecipitation was performed with a specific anti-FLAG-tag antibody. Precipitated proteins were analyzed by SDS–PAGE, followed by western blotting with an anti-Myc-tag antibody. Aliquots corresponding to 1% of the used cell lysates were analyzed in parallel (Input 1%). ( B ) GST or the GST fusion protein of NCoA-1 PAS-B domain (aa 260–370) were bound to glutathione Sepharose beads and incubated with a [ 35 S]methionine-labeled fragment of the STAT6-TAD (aa 792–847) in absence or presence of 45 µM of a purified NCoA-1 CID/AD1 peptide (aa 901–970). After washing and elution, precipitated proteins were analyzed by SDS–PAGE and fluorography. The CID/AD1 fragment was bacterially expressed, digested and purified as described in Materials and Methods. An aliquot representing 10% of the in vitro transcribed/translated fragment was analyzed in parallel (Input). A quarter of each binding reaction was separately analyzed by SDS–PAGE and Coomassie staining in order to prove same amounts of GST or GST fusion proteins. ( C ) Quantification of bound STAT6-TAD fragment to GST or GST fusion protein of the NCoA-1 PAS-B domain in absence or presence of the CID/AD1 peptide. The relative radioactivity (cpm; counts per minute) was determined by normalization against 10% of the in vitro transcribed/translated protein. The average of three independent experiments with standard deviation is shown.
Figure Legend Snippet: Overexpression of the NCoA PAS-B domain or competition with the CID/AD1 inhibits contact of NCoA-1 to CBP or the STAT6-TAD. ( A ) 293T cells were transiently transfected with expression vectors encoding a FLAG-tagged CBP fragment (aa 2058–2130) or the empty FLAG-tag vector (pMX-FLAG) (each 1 µg), a 6× Myc-tagged CID/AD1 fragment of NCoA-1 (aa 804–1032) (0,1 µg) along with a vector containing a YFP fusion protein encoding NCoA-1 aa 213–462 or CFP as a control (each 3 µg). Whole cell extracts were prepared and co-immunoprecipitation was performed with a specific anti-FLAG-tag antibody. Precipitated proteins were analyzed by SDS–PAGE, followed by western blotting with an anti-Myc-tag antibody. Aliquots corresponding to 1% of the used cell lysates were analyzed in parallel (Input 1%). ( B ) GST or the GST fusion protein of NCoA-1 PAS-B domain (aa 260–370) were bound to glutathione Sepharose beads and incubated with a [ 35 S]methionine-labeled fragment of the STAT6-TAD (aa 792–847) in absence or presence of 45 µM of a purified NCoA-1 CID/AD1 peptide (aa 901–970). After washing and elution, precipitated proteins were analyzed by SDS–PAGE and fluorography. The CID/AD1 fragment was bacterially expressed, digested and purified as described in Materials and Methods. An aliquot representing 10% of the in vitro transcribed/translated fragment was analyzed in parallel (Input). A quarter of each binding reaction was separately analyzed by SDS–PAGE and Coomassie staining in order to prove same amounts of GST or GST fusion proteins. ( C ) Quantification of bound STAT6-TAD fragment to GST or GST fusion protein of the NCoA-1 PAS-B domain in absence or presence of the CID/AD1 peptide. The relative radioactivity (cpm; counts per minute) was determined by normalization against 10% of the in vitro transcribed/translated protein. The average of three independent experiments with standard deviation is shown.

Techniques Used: Over Expression, Transfection, Expressing, FLAG-tag, Plasmid Preparation, Immunoprecipitation, SDS Page, Western Blot, Incubation, Labeling, Purification, In Vitro, Binding Assay, Staining, Radioactivity, Standard Deviation

LXXLL motifs differentially contribute to the interaction of the PAS-B domains and the CID/AD1 of NCoA-1. ( A ) Sequence alignment of the fragments in the CID/AD1 of NCoA-1 and NCoA-3. LXXLL motifs are printed in bold. The regions of NCoA-1 and NCoA-3 which mediate the interaction with CBP are indicated in boxes (NCoA-1 aa 926–960 and NCoA-3 aa 1045–1091). Terminal amino acids of each construct are shown. Sequences of NCoA-1 and NCoA-3 correspond to the protein ID's AAI11534 and AAC51677, respectively. ( B ) Fragments of NCoA-1 comprising amino acids 901–970, 901–937 or 938–970 and NCoA-3 comprising amino acids 1022–1092 of the CID/AD1 were in vitro transcribed/translated and [ 35 S]methionine-labeled. The fragments were incubated with GST alone or GST fusion proteins of the PAS-B domains of all three NCoA family members or a fusion protein of the CBP NCoA interacting region (aa 2058–2130) bound to glutathione Sepharose. Precipitated proteins were analyzed by SDS–PAGE and fluorography. 10% of radioactive-labeled material used for interaction assays was analyzed in parallel (Input). ( C ) Structure of NCoA-1. Different functional domains (grey boxes) and LXXLL motifs (black bars) are indicated. Peptides derived from the CID/AD1 used in competition experiments are shown. The sequences representing LXXLL motif 4 and motif 5 are printed in bold. GST pulldown assays were performed as described in B, with the NCoA PAS-B domains and the [ 35 S]methionine-labeled fragment of the NCoA-1 CID/AD1 in absence or presence of 280, 28 or 2.8 µM of each peptide. ( D ) Schematic representation of the NCoA-1 CID/AD1 sequence. LXXLL motifs 4 and 5 are printed in bold. Point mutations of leucines to alanines are depicted for motif 4 (MutM4), motif 5 (MutM5) and both motifs (MutM4+5). ( E ) Experiments were performed as described in B with the radioactive-labeled fragments containing residues 901–970 of wild type or mutant (MutM4, MutM5 or MutM4+5) NCoA-1. ( F ) Quantification of bound radioactive-labeled fragments of wild type or mutant NCoA-1 CID/AD1 (aa 901–970) to GST and GST fusion proteins. Relative radioactivity (cpm; counts per minute) was determined by normalization against an aliquot representing 10% of the radioactive-labeled material. The average of three independent experiments with standard deviation is shown. ( G ) Chemical shift perturbation plot on NCoA-1 sequence upon peptides binding. 1 H- 15 N HSQC titration data for the addition of LXXLL peptides to the 15 N -NCoA-1 PAS-B domain Δδ values (calculated as described in Materials and Methods) generated upon motif 4 (blue bars), motif 5 (red bars) and STAT6 peptide (green bars) binding are plotted against the NCoA-1 PAS-B domain sequence.
Figure Legend Snippet: LXXLL motifs differentially contribute to the interaction of the PAS-B domains and the CID/AD1 of NCoA-1. ( A ) Sequence alignment of the fragments in the CID/AD1 of NCoA-1 and NCoA-3. LXXLL motifs are printed in bold. The regions of NCoA-1 and NCoA-3 which mediate the interaction with CBP are indicated in boxes (NCoA-1 aa 926–960 and NCoA-3 aa 1045–1091). Terminal amino acids of each construct are shown. Sequences of NCoA-1 and NCoA-3 correspond to the protein ID's AAI11534 and AAC51677, respectively. ( B ) Fragments of NCoA-1 comprising amino acids 901–970, 901–937 or 938–970 and NCoA-3 comprising amino acids 1022–1092 of the CID/AD1 were in vitro transcribed/translated and [ 35 S]methionine-labeled. The fragments were incubated with GST alone or GST fusion proteins of the PAS-B domains of all three NCoA family members or a fusion protein of the CBP NCoA interacting region (aa 2058–2130) bound to glutathione Sepharose. Precipitated proteins were analyzed by SDS–PAGE and fluorography. 10% of radioactive-labeled material used for interaction assays was analyzed in parallel (Input). ( C ) Structure of NCoA-1. Different functional domains (grey boxes) and LXXLL motifs (black bars) are indicated. Peptides derived from the CID/AD1 used in competition experiments are shown. The sequences representing LXXLL motif 4 and motif 5 are printed in bold. GST pulldown assays were performed as described in B, with the NCoA PAS-B domains and the [ 35 S]methionine-labeled fragment of the NCoA-1 CID/AD1 in absence or presence of 280, 28 or 2.8 µM of each peptide. ( D ) Schematic representation of the NCoA-1 CID/AD1 sequence. LXXLL motifs 4 and 5 are printed in bold. Point mutations of leucines to alanines are depicted for motif 4 (MutM4), motif 5 (MutM5) and both motifs (MutM4+5). ( E ) Experiments were performed as described in B with the radioactive-labeled fragments containing residues 901–970 of wild type or mutant (MutM4, MutM5 or MutM4+5) NCoA-1. ( F ) Quantification of bound radioactive-labeled fragments of wild type or mutant NCoA-1 CID/AD1 (aa 901–970) to GST and GST fusion proteins. Relative radioactivity (cpm; counts per minute) was determined by normalization against an aliquot representing 10% of the radioactive-labeled material. The average of three independent experiments with standard deviation is shown. ( G ) Chemical shift perturbation plot on NCoA-1 sequence upon peptides binding. 1 H- 15 N HSQC titration data for the addition of LXXLL peptides to the 15 N -NCoA-1 PAS-B domain Δδ values (calculated as described in Materials and Methods) generated upon motif 4 (blue bars), motif 5 (red bars) and STAT6 peptide (green bars) binding are plotted against the NCoA-1 PAS-B domain sequence.

Techniques Used: Sequencing, Construct, In Vitro, Labeling, Incubation, SDS Page, Functional Assay, Derivative Assay, Mutagenesis, Radioactivity, Standard Deviation, Binding Assay, Titration, Generated

Interaction of NCoA proteins through the PAS-B domains and CID/AD1. ( A ) Schematic representation of NCoA-1, different NCoA-1 constructs and the STAT6-TAD fragment. bHLH domain, PAS domain, NID [three LXXLL motifs (black bars)], CID/AD1 (two LXXLL motifs) and AD2 (one LXXLL motif) are indicated. The STAT6 peptide comprises the NCoA-1 interacting LXXLL motif (black bar). The terminal amino acids of each construct are shown. The NCoA-1 fragments representing different regions containing LXXLL motifs were in vitro transcribed/translated and [ 35 S]methionine-labeled. A STAT6 fragment which contains the NCoA-1 interacting LXXLL motif was used as a control. After incubation with GST or GST fusion proteins of NCoA-1, NCoA-2 or NCoA-3 PAS-B domains, bound to glutathione Sepharose beads, proteins were eluted and analyzed by SDS–PAGE and fluorography. As an input control 10% of the radioactive-labeled proteins were analyzed in parallel. ( B ) Experiments were performed with the radioactive-labeled full-length NCoA-1, NCoA-2 and NCoA-3 as described before. ( C ) 293T cells were transiently transfected with expression vectors encoding either a YFP fusion protein of NCoA-1 aa 213–462, containing the PAS-B domain, or CFP and full-length NCoA-1 (each 3 µg). Cell extracts were prepared and immunoprecipitation (IP) was performed with an anti-GFP antibody, recognizing CFP and YFP. Proteins were analyzed by SDS–PAGE and western blotting with an anti-NCoA-1 antibody. As a control 1% of the cell lysates were analyzed in parallel (Input 1%). ( D ) The GST pulldown assay was performed with the GST fusion protein comprising the aminoterminal region of NCoA-1 (aa 1–370) and the [ 35 S]methionine-labeled fragments of NCoA-1 containing the aminoterminal region or the CID/AD1 (aa 804–1032).
Figure Legend Snippet: Interaction of NCoA proteins through the PAS-B domains and CID/AD1. ( A ) Schematic representation of NCoA-1, different NCoA-1 constructs and the STAT6-TAD fragment. bHLH domain, PAS domain, NID [three LXXLL motifs (black bars)], CID/AD1 (two LXXLL motifs) and AD2 (one LXXLL motif) are indicated. The STAT6 peptide comprises the NCoA-1 interacting LXXLL motif (black bar). The terminal amino acids of each construct are shown. The NCoA-1 fragments representing different regions containing LXXLL motifs were in vitro transcribed/translated and [ 35 S]methionine-labeled. A STAT6 fragment which contains the NCoA-1 interacting LXXLL motif was used as a control. After incubation with GST or GST fusion proteins of NCoA-1, NCoA-2 or NCoA-3 PAS-B domains, bound to glutathione Sepharose beads, proteins were eluted and analyzed by SDS–PAGE and fluorography. As an input control 10% of the radioactive-labeled proteins were analyzed in parallel. ( B ) Experiments were performed with the radioactive-labeled full-length NCoA-1, NCoA-2 and NCoA-3 as described before. ( C ) 293T cells were transiently transfected with expression vectors encoding either a YFP fusion protein of NCoA-1 aa 213–462, containing the PAS-B domain, or CFP and full-length NCoA-1 (each 3 µg). Cell extracts were prepared and immunoprecipitation (IP) was performed with an anti-GFP antibody, recognizing CFP and YFP. Proteins were analyzed by SDS–PAGE and western blotting with an anti-NCoA-1 antibody. As a control 1% of the cell lysates were analyzed in parallel (Input 1%). ( D ) The GST pulldown assay was performed with the GST fusion protein comprising the aminoterminal region of NCoA-1 (aa 1–370) and the [ 35 S]methionine-labeled fragments of NCoA-1 containing the aminoterminal region or the CID/AD1 (aa 804–1032).

Techniques Used: Construct, In Vitro, Labeling, Incubation, SDS Page, Transfection, Expressing, Immunoprecipitation, Western Blot, GST Pulldown Assay

22) Product Images from "C/EBPBeta and Elk-1 synergistically transactivate the c-fos serum response element"

Article Title: C/EBPBeta and Elk-1 synergistically transactivate the c-fos serum response element

Journal: BMC Cell Biology

doi: 10.1186/1471-2121-1-2

The C-terminal domain of C/EBPβ interacts with Elk-1. Radiolabeled Elk-1 was prepared by in vitro transcription and translation of CMV-Elk-1 expression construct and then incubated with equivalent amounts of GST, GST-p35-C/EBPβ, or GST-p20-C/EBPβ protein immobilized on glutathione-Sepharose. Proteins eluted from the GST-p20-C/EBPβ beads (lane 2), the GST-p35-C/EBPβ beads (lane 3) or GST beads (lanes 4) were resolved on an SDS-10% polyacrylamide gel and detected by autoradiography. Lane 1 contains one-half of the amount of the radiolabeled proteins initially mixed with the beads.
Figure Legend Snippet: The C-terminal domain of C/EBPβ interacts with Elk-1. Radiolabeled Elk-1 was prepared by in vitro transcription and translation of CMV-Elk-1 expression construct and then incubated with equivalent amounts of GST, GST-p35-C/EBPβ, or GST-p20-C/EBPβ protein immobilized on glutathione-Sepharose. Proteins eluted from the GST-p20-C/EBPβ beads (lane 2), the GST-p35-C/EBPβ beads (lane 3) or GST beads (lanes 4) were resolved on an SDS-10% polyacrylamide gel and detected by autoradiography. Lane 1 contains one-half of the amount of the radiolabeled proteins initially mixed with the beads.

Techniques Used: In Vitro, Expressing, Construct, Incubation, Autoradiography

Elk-1 binds to GST-p35-C/EBPβ in vitro . Radiolabeled Elk-1 was prepared by in vitro transcription and translation of CMV-Elk-1 expression construct and then incubated with equivalent amounts of GST or GST-p35-C/EBPβ protein immobilized on glutathione-Sepharose. Proteins eluted from the GST-p35-C/EBPβ beads (lanes 2) or GST beads (lanes 3) by boiling in Laemmli sample buffer were resolved on an SDS-10% polyacrylamide gel and detected by autoradiography. Lane 1 contains one-fourth of the amount of the radiolabeled proteins initially mixed with the beads.
Figure Legend Snippet: Elk-1 binds to GST-p35-C/EBPβ in vitro . Radiolabeled Elk-1 was prepared by in vitro transcription and translation of CMV-Elk-1 expression construct and then incubated with equivalent amounts of GST or GST-p35-C/EBPβ protein immobilized on glutathione-Sepharose. Proteins eluted from the GST-p35-C/EBPβ beads (lanes 2) or GST beads (lanes 3) by boiling in Laemmli sample buffer were resolved on an SDS-10% polyacrylamide gel and detected by autoradiography. Lane 1 contains one-fourth of the amount of the radiolabeled proteins initially mixed with the beads.

Techniques Used: In Vitro, Expressing, Construct, Incubation, Autoradiography

Elk-1 coimmunoprecipitates with p35-C/EBPβ, but only in the presence of activated Ras. COS-7 cells were transfected with 10 μg of pCDNA3.1/His-p35-C/EBPβ (lanes 1, 2, 5, 6) and 10 μg of pCMV-Elk-1 (lanes 3, 4, 5, 6) in the presence and absence of 2 μg of pCMV-Ras.V12 as indicated. Cells were harvested 40 h post-transfection, and whole cell lysates were incubated with T7tag Ab-agarose beads, followed by immunoblotting of precipitated proteins with Elk-1 Ab.
Figure Legend Snippet: Elk-1 coimmunoprecipitates with p35-C/EBPβ, but only in the presence of activated Ras. COS-7 cells were transfected with 10 μg of pCDNA3.1/His-p35-C/EBPβ (lanes 1, 2, 5, 6) and 10 μg of pCMV-Elk-1 (lanes 3, 4, 5, 6) in the presence and absence of 2 μg of pCMV-Ras.V12 as indicated. Cells were harvested 40 h post-transfection, and whole cell lysates were incubated with T7tag Ab-agarose beads, followed by immunoblotting of precipitated proteins with Elk-1 Ab.

Techniques Used: Transfection, Incubation

The A-box of Elk-1 is necessary for interaction with p35-C/EBPβ in vitro . A) Schematic representation of Elk-1 wild-type and deletion mutants. B) Radiolabeled Elk-1(wt), Elk-1(1-209), Elk-1(1-140), and Elk-1(89-428) were prepared by in vitro transcription and translation of their respective expression constructs (see Mat. and Meth.). The proteins were incubated with equivalent amounts of GST or GST-p35-C/EBPβ protein immobilized on glutathione-Sepharose. Proteins eluted from the GST beads (lanes 2) or GST-p35-C/EBPβ beads (lanes 3) were resolved on an SDS-10% polyacrylamide gel and detected by autoradiography. Lane 1 contains one-half of the amount of the radiolabeled proteins initially mixed with the beads.
Figure Legend Snippet: The A-box of Elk-1 is necessary for interaction with p35-C/EBPβ in vitro . A) Schematic representation of Elk-1 wild-type and deletion mutants. B) Radiolabeled Elk-1(wt), Elk-1(1-209), Elk-1(1-140), and Elk-1(89-428) were prepared by in vitro transcription and translation of their respective expression constructs (see Mat. and Meth.). The proteins were incubated with equivalent amounts of GST or GST-p35-C/EBPβ protein immobilized on glutathione-Sepharose. Proteins eluted from the GST beads (lanes 2) or GST-p35-C/EBPβ beads (lanes 3) were resolved on an SDS-10% polyacrylamide gel and detected by autoradiography. Lane 1 contains one-half of the amount of the radiolabeled proteins initially mixed with the beads.

Techniques Used: In Vitro, Expressing, Construct, Incubation, Autoradiography

23) Product Images from "Phosphorylation of Ku70 subunit by cell cycle kinases modulates the replication related function of Ku heterodimer"

Article Title: Phosphorylation of Ku70 subunit by cell cycle kinases modulates the replication related function of Ku heterodimer

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw622

Ku70 interacts with cyclin B1. ( A ) Glutathione Sepharose beads bound to GST or GST-cyclin B1 (expressed in insect cells) were incubated with bacterially expressed and purified Ku70 (lanes 1 and 2), insect cell extract containing over-expressed Ku70 (lanes 3 and 4) or HEK293 cell extract (lanes 5 and 6). The pulled-down complexes and the supernatant were analyzed by immunoblotting (IB) with antibodies against Ku70 (Interaction and Input) and GST (Input). ( B ) GST or GST-Ku70 were expressed in HeLa cells and pulled down from the cell-extract with glutathione beads. The presence or absence of cyclin B1 in the extract (lanes 1 and 2) and the pulled-down complexes (lanes 3 and 4) was tested by anti-cyclin B1 immunoblotting. The expression of GST and GST-Ku70 and their presence in the beads were also checked by anti-GST immunoblotting.
Figure Legend Snippet: Ku70 interacts with cyclin B1. ( A ) Glutathione Sepharose beads bound to GST or GST-cyclin B1 (expressed in insect cells) were incubated with bacterially expressed and purified Ku70 (lanes 1 and 2), insect cell extract containing over-expressed Ku70 (lanes 3 and 4) or HEK293 cell extract (lanes 5 and 6). The pulled-down complexes and the supernatant were analyzed by immunoblotting (IB) with antibodies against Ku70 (Interaction and Input) and GST (Input). ( B ) GST or GST-Ku70 were expressed in HeLa cells and pulled down from the cell-extract with glutathione beads. The presence or absence of cyclin B1 in the extract (lanes 1 and 2) and the pulled-down complexes (lanes 3 and 4) was tested by anti-cyclin B1 immunoblotting. The expression of GST and GST-Ku70 and their presence in the beads were also checked by anti-GST immunoblotting.

Techniques Used: Incubation, Purification, Expressing

Ku70 is phosphorylated by cyclin-Cdks in a Cy-motif dependent manner. ( A ) The wild type and mutant (Thr to Ala, as indicated) Ku70 proteins were used in [γ- 32 P]ATP-based kinase assays with the cyclin-Cdks as marked. For each set of assays, the presence of equal amounts of Ku70 protein was ascertained by Coomassie blue staining of the reaction mixtures. The radioactive Ku70 bands due to incorporation of 32 P are indicated. ( B ) GST or GST-cyclin B1 bound to glutathione sepharose beads were incubated with purified Ku70 protein either in absence of any peptide (lanes 1 and 2), or in presence of 150 μM competitor SM100 peptide (lane 3) or control AM100 peptide (lane 4). The pulled-down complexes were analyzed by immunoblotting with antibodies against Ku70 (interaction) and GST (input). The presence of equivalent amounts of Ku70 protein in interaction mixtures were checked by anti-Ku70 immunoblotting (input). ( C ) HsKu70 was used as substrate in [γ- 32 P]ATP based kinase assay with cyclin B1–Cdk1 in the absence or presence of 150 μM of the indicated peptides.
Figure Legend Snippet: Ku70 is phosphorylated by cyclin-Cdks in a Cy-motif dependent manner. ( A ) The wild type and mutant (Thr to Ala, as indicated) Ku70 proteins were used in [γ- 32 P]ATP-based kinase assays with the cyclin-Cdks as marked. For each set of assays, the presence of equal amounts of Ku70 protein was ascertained by Coomassie blue staining of the reaction mixtures. The radioactive Ku70 bands due to incorporation of 32 P are indicated. ( B ) GST or GST-cyclin B1 bound to glutathione sepharose beads were incubated with purified Ku70 protein either in absence of any peptide (lanes 1 and 2), or in presence of 150 μM competitor SM100 peptide (lane 3) or control AM100 peptide (lane 4). The pulled-down complexes were analyzed by immunoblotting with antibodies against Ku70 (interaction) and GST (input). The presence of equivalent amounts of Ku70 protein in interaction mixtures were checked by anti-Ku70 immunoblotting (input). ( C ) HsKu70 was used as substrate in [γ- 32 P]ATP based kinase assay with cyclin B1–Cdk1 in the absence or presence of 150 μM of the indicated peptides.

Techniques Used: Mutagenesis, Staining, Incubation, Purification, Kinase Assay

Cyclin B1 interacts with Ku70 in Ku-heterodimer in vitro and in vivo . ( A ) Varying quantities of 6 His-Ku70 monomer (lanes 1–4) and 6 His-Ku70/SBP-Ku80 dimer (lanes 5–8), purified from insect cell expression system, were incubated with glutathione agarose beads bound to GST or GST-cyclin B1 (expressed in insect cells). The pulled-down complexes were analyzed by anti-Ku70 and anti-SBP tag (for Ku80) immunoblotting (Interaction). The presence of Ku70 and Ku80, as appropriate, in the reaction mixtures was checked by indicated immunoblotting (Input panels). The presence of GST or GST-cyclin B1 was also tested by anti-GST immunoblotting. ( B ) Immunoprecipitation with antibodies against cyclin B1 and Ku70 were carried out from the extract of HeLa or HEK293 cells arrested at mitosis by nocodazole and the presence of Ku70, Ku80 or Cdc2 in the immunoprecipitates were determined by immunoblotting with appropriate antibodies as indicated.
Figure Legend Snippet: Cyclin B1 interacts with Ku70 in Ku-heterodimer in vitro and in vivo . ( A ) Varying quantities of 6 His-Ku70 monomer (lanes 1–4) and 6 His-Ku70/SBP-Ku80 dimer (lanes 5–8), purified from insect cell expression system, were incubated with glutathione agarose beads bound to GST or GST-cyclin B1 (expressed in insect cells). The pulled-down complexes were analyzed by anti-Ku70 and anti-SBP tag (for Ku80) immunoblotting (Interaction). The presence of Ku70 and Ku80, as appropriate, in the reaction mixtures was checked by indicated immunoblotting (Input panels). The presence of GST or GST-cyclin B1 was also tested by anti-GST immunoblotting. ( B ) Immunoprecipitation with antibodies against cyclin B1 and Ku70 were carried out from the extract of HeLa or HEK293 cells arrested at mitosis by nocodazole and the presence of Ku70, Ku80 or Cdc2 in the immunoprecipitates were determined by immunoblotting with appropriate antibodies as indicated.

Techniques Used: In Vitro, In Vivo, Purification, Expressing, Incubation, Immunoprecipitation

24) Product Images from "Ponsin/SH3P12: An l-Afadin- and Vinculin-binding Protein Localized at Cell-Cell and Cell-Matrix Adherens Junctions "

Article Title: Ponsin/SH3P12: An l-Afadin- and Vinculin-binding Protein Localized at Cell-Cell and Cell-Matrix Adherens Junctions

Journal: The Journal of Cell Biology

doi:

Inability of ponsin to form a ternary complex with l-afadin and vinculin. (A) Immunoprecipitation from COS7 cells. Myc– l-afadin (the full length), FLAG–ponsin-2 (full length), and Myc–vinculin-C (the COOH-terminal half) were expressed in COS7 cells in various combinations by transfection with pCMV-Myc–l-afadin, pFLAG-CMV2–ponsin-2, and pCMV-Myc–vinculin-C, respectively. Each cell extract was subjected to immunoprecipitation with the anti–Myc or anti–FLAG antibody. The precipitate was then subjected to SDS-PAGE (8% polyacrylamide gel), followed by Western blot analysis using the anti–Myc, anti–FLAG, anti–l-afadin, or anti-vinculin antibody. (A1) COS7 cells expressing FLAG–ponsin-2 alone, (A2) COS7 cells expressing both FLAG–ponsin-2 and Myc–vinculin-C, (A3) COS7 cells expressing both Myc–l-afadin and FLAG–ponsin-2. (IP) Immunoprecipitation. (B) Affinity chromatography using ponsin-immobilized beads. Various doses of His6–l-afadin-C199 (the COOH-terminal 199 aa region including the third proline-rich region) and MBP–vinculin-C (the COOH-terminal half) were mixed and incubated with GST–ponsin-2-C (the COOH-terminal half) immobilized on glutathione-Sepharose beads. Each eluate was subjected to SDS-PAGE (15% polyacrylamide gel), followed by protein staining with Coomassie brilliant blue. The results are representative of three independent experiments.
Figure Legend Snippet: Inability of ponsin to form a ternary complex with l-afadin and vinculin. (A) Immunoprecipitation from COS7 cells. Myc– l-afadin (the full length), FLAG–ponsin-2 (full length), and Myc–vinculin-C (the COOH-terminal half) were expressed in COS7 cells in various combinations by transfection with pCMV-Myc–l-afadin, pFLAG-CMV2–ponsin-2, and pCMV-Myc–vinculin-C, respectively. Each cell extract was subjected to immunoprecipitation with the anti–Myc or anti–FLAG antibody. The precipitate was then subjected to SDS-PAGE (8% polyacrylamide gel), followed by Western blot analysis using the anti–Myc, anti–FLAG, anti–l-afadin, or anti-vinculin antibody. (A1) COS7 cells expressing FLAG–ponsin-2 alone, (A2) COS7 cells expressing both FLAG–ponsin-2 and Myc–vinculin-C, (A3) COS7 cells expressing both Myc–l-afadin and FLAG–ponsin-2. (IP) Immunoprecipitation. (B) Affinity chromatography using ponsin-immobilized beads. Various doses of His6–l-afadin-C199 (the COOH-terminal 199 aa region including the third proline-rich region) and MBP–vinculin-C (the COOH-terminal half) were mixed and incubated with GST–ponsin-2-C (the COOH-terminal half) immobilized on glutathione-Sepharose beads. Each eluate was subjected to SDS-PAGE (15% polyacrylamide gel), followed by protein staining with Coomassie brilliant blue. The results are representative of three independent experiments.

Techniques Used: Immunoprecipitation, Transfection, SDS Page, Western Blot, Expressing, Affinity Chromatography, Incubation, Staining

Binding regions of ponsin and vinculin. (A) Blot overlay. (A1) 35 S-Labeled vinculin blot overlay. Various GST-fused proteins of ponsin-2 (5 pmol each) were subjected to SDS-PAGE (8% polyacrylamide gel), followed by 35 S-labeled vinculin blot overlay. (A2) 35 S- Labeled ponsin-2 blot overlay. Full-length native vinculin and various recombinant proteins of vinculin (5 pmol each) were subjected to SDS-PAGE (8 and 12% discontinuous polyacrylamide gel), followed by 35 S- labeled ponsin blot overlay. (B) Affinity chromatography. (B1) Binding region of ponsin. MBP– vinculin-C (the COOH-terminal half) was incubated with various GST-fused proteins of ponsin-2 immobilized on glutathione-Sepharose beads. Each eluate was subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blot analysis using the anti-vinculin antibody. (B2) Binding region of vinculin. MBP–vinculin-C or MBP alone was incubated with GST–ponsin-2-C (the COOH-terminal half) or GST alone immobilized on glutathione-Sepharose beads. Each original sample and eluate were subjected to SDS-PAGE (12% polyacrylamide gel), followed by protein staining with Coomassie brilliant blue. (F) GST– ponsin-2-F (the full length), (N) GST–ponsin-2-N (the NH 2 -terminal half), (C) GST–ponsin-2-C, [SH3(1+2)] GST–ponsin-2-SH3(1+2) (the first and second SH3 domains), [SH3(2+3)] GST–ponsin-2-SH3(2+3) (the second and third SH3 domains), [SH3(1)] GST–ponsin-2-SH3(1) (the first SH3 domain), [SH3(2)] GST–ponsin-2-SH3(2) (the second SH3 domain), [SH3(3)] GST–ponsin-2-SH3(3) (the third SH3 domain), [PR] the proline-rich region of vinculin (aa 837–878), (1) Myc–vinculin-1 (the NH 2 -terminal region, aa 1–800), (2) Myc–vinculin-2 (the COOH-terminal region, aa 801–1066); (P) GST–vinculin-P (the proline-rich region), and (C-ΔP) GST–vinculin-C-ΔP (the COOH-terminal region lacking the proline-rich region). The results are representative of three independent experiments.
Figure Legend Snippet: Binding regions of ponsin and vinculin. (A) Blot overlay. (A1) 35 S-Labeled vinculin blot overlay. Various GST-fused proteins of ponsin-2 (5 pmol each) were subjected to SDS-PAGE (8% polyacrylamide gel), followed by 35 S-labeled vinculin blot overlay. (A2) 35 S- Labeled ponsin-2 blot overlay. Full-length native vinculin and various recombinant proteins of vinculin (5 pmol each) were subjected to SDS-PAGE (8 and 12% discontinuous polyacrylamide gel), followed by 35 S- labeled ponsin blot overlay. (B) Affinity chromatography. (B1) Binding region of ponsin. MBP– vinculin-C (the COOH-terminal half) was incubated with various GST-fused proteins of ponsin-2 immobilized on glutathione-Sepharose beads. Each eluate was subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blot analysis using the anti-vinculin antibody. (B2) Binding region of vinculin. MBP–vinculin-C or MBP alone was incubated with GST–ponsin-2-C (the COOH-terminal half) or GST alone immobilized on glutathione-Sepharose beads. Each original sample and eluate were subjected to SDS-PAGE (12% polyacrylamide gel), followed by protein staining with Coomassie brilliant blue. (F) GST– ponsin-2-F (the full length), (N) GST–ponsin-2-N (the NH 2 -terminal half), (C) GST–ponsin-2-C, [SH3(1+2)] GST–ponsin-2-SH3(1+2) (the first and second SH3 domains), [SH3(2+3)] GST–ponsin-2-SH3(2+3) (the second and third SH3 domains), [SH3(1)] GST–ponsin-2-SH3(1) (the first SH3 domain), [SH3(2)] GST–ponsin-2-SH3(2) (the second SH3 domain), [SH3(3)] GST–ponsin-2-SH3(3) (the third SH3 domain), [PR] the proline-rich region of vinculin (aa 837–878), (1) Myc–vinculin-1 (the NH 2 -terminal region, aa 1–800), (2) Myc–vinculin-2 (the COOH-terminal region, aa 801–1066); (P) GST–vinculin-P (the proline-rich region), and (C-ΔP) GST–vinculin-C-ΔP (the COOH-terminal region lacking the proline-rich region). The results are representative of three independent experiments.

Techniques Used: Binding Assay, Labeling, SDS Page, Recombinant, Affinity Chromatography, Incubation, Western Blot, Staining

Binding of l-afadin to ponsin in vivo. (A) Immunoprecipitation analysis of l-afadin and ponsin-2. FLAG–ponsin-2 (the full length) and Myc–l-afadin (the full length) were coexpressed in COS7 cells in various combinations by transfection with pFLAG-CMV2–ponsin-2 and pCMV-Myc–l-afadin, respectively. Each cell extract was subjected to immunoprecipitation with the anti–FLAG or anti–Myc antibody. The precipitate was then subjected to SDS-PAGE (8% polyacrylamide gel), followed by Western blot analysis using the anti–FLAG or anti–Myc antibody, or by protein staining with Coomassie brilliant blue. (A1) Western blot analysis, and (A2) protein staining. (IP) Immunoprecipitation, and (CBB) staining with Coomassie brilliant blue. (B) Immunoprecipitation analysis of s-afadin and ponsin-2. Both FLAG–ponsin-2 and tag-free l-afadin (the full length) or both FLAG–ponsin-2 and tag-free s-afadin (the full length) were coexpressed in COS7 cells by transfection with both pFLAG-CMV2– ponsin-2 and pCMV5–l-afadin or both pFLAG-CMV2–ponsin-2 and pCMV5–s-afadin, respectively. Each cell extract was incubated with the anti–FLAG antibody and protein G–Sepharose beads, followed by centrifugation. The beads and the supernatant were separately subjected to SDS-PAGE (8% polyacrylamide gel), followed by Western blot analysis using the anti–l- and s-afadin antibody. (Sup) supernatant. The results are representative of three independent experiments.
Figure Legend Snippet: Binding of l-afadin to ponsin in vivo. (A) Immunoprecipitation analysis of l-afadin and ponsin-2. FLAG–ponsin-2 (the full length) and Myc–l-afadin (the full length) were coexpressed in COS7 cells in various combinations by transfection with pFLAG-CMV2–ponsin-2 and pCMV-Myc–l-afadin, respectively. Each cell extract was subjected to immunoprecipitation with the anti–FLAG or anti–Myc antibody. The precipitate was then subjected to SDS-PAGE (8% polyacrylamide gel), followed by Western blot analysis using the anti–FLAG or anti–Myc antibody, or by protein staining with Coomassie brilliant blue. (A1) Western blot analysis, and (A2) protein staining. (IP) Immunoprecipitation, and (CBB) staining with Coomassie brilliant blue. (B) Immunoprecipitation analysis of s-afadin and ponsin-2. Both FLAG–ponsin-2 and tag-free l-afadin (the full length) or both FLAG–ponsin-2 and tag-free s-afadin (the full length) were coexpressed in COS7 cells by transfection with both pFLAG-CMV2– ponsin-2 and pCMV5–l-afadin or both pFLAG-CMV2–ponsin-2 and pCMV5–s-afadin, respectively. Each cell extract was incubated with the anti–FLAG antibody and protein G–Sepharose beads, followed by centrifugation. The beads and the supernatant were separately subjected to SDS-PAGE (8% polyacrylamide gel), followed by Western blot analysis using the anti–l- and s-afadin antibody. (Sup) supernatant. The results are representative of three independent experiments.

Techniques Used: Binding Assay, In Vivo, Immunoprecipitation, Transfection, SDS Page, Western Blot, Staining, Incubation, Centrifugation

Binding regions of l-afadin and ponsin. (A) Blot overlay. (A1) 35 S-Labeled ponsin-2 blot overlay. Myc–l-Afadin (the full length), Myc–s-afadin (the full length), His6–l-afadin-C199 (the COOH-terminal 199 aa region including the third proline-rich region), and His6– l-afadin-C132 (the COOH-terminal 132 aa region lacking the aa residues [PPLP] of the third proline-rich domain) (5 pmol each) were subjected to SDS-PAGE (8 and 12% discontinuous polyacrylamide gel), followed by 35 S-labeled ponsin-2 blot overlay. (A2) 35 S-Labeled l-afadin blot overlay. Various GST-fused proteins of ponsin-2 (5 pmol each) were subjected to SDS-PAGE (8% polyacrylamide gel), followed by 35 S-labeled l-afadin blot overlay. (B) Affinity chromatography. (B1) Binding region of l-afadin. His6–l-Afadin-C199 or His6–l-afadin-C132 was incubated with GST–ponsin-2-C (the COOH-terminal half) or GST alone immobilized on glutathione-Sepharose beads. Each original sample and eluate were subjected to SDS-PAGE (15% polyacrylamide gel), followed by protein staining with Coomassie brilliant blue. (B2) Binding region of ponsin. His6– l-afadin-C199 was incubated with various GST-fused proteins of ponsin-2 immobilized on glutathione-Sepharose beads. Each eluate was subjected to SDS-PAGE (12% polyacrylamide gel), followed by Western blot analysis using the anti–l-afadin antibody. [PR(1)] the first proline-rich region (aa 1219–1229), [PR(2)] the second proline-rich region (aa 1372–1399), [PR(3)] the third proline-rich region (aa 1691–1713), (C199) His6–l-afadin-C199, (C132) His6–l-afadin-C132, (F) GST–ponsin-2-F (the full length), (N) GST–ponsin-2-N (the NH 2 -terminal half), (C) GST–ponsin-2-C, [SH3(1+2)] GST– ponsin-2-SH3(1+2) (the first and second SH3 domains), [SH3(2+3)] GST–ponsin-2-SH3(2+3) (the second and third SH3 domains), [SH3(1)] GST–ponsin-2-SH3(1) (the first SH3 domain), [SH3(2)] GST–ponsin-2-SH3(2) (the second SH3 domain), and [SH3(3)] GST– ponsin-2-SH3(3) (the third SH3 domain). The results are representative of three independent experiments.
Figure Legend Snippet: Binding regions of l-afadin and ponsin. (A) Blot overlay. (A1) 35 S-Labeled ponsin-2 blot overlay. Myc–l-Afadin (the full length), Myc–s-afadin (the full length), His6–l-afadin-C199 (the COOH-terminal 199 aa region including the third proline-rich region), and His6– l-afadin-C132 (the COOH-terminal 132 aa region lacking the aa residues [PPLP] of the third proline-rich domain) (5 pmol each) were subjected to SDS-PAGE (8 and 12% discontinuous polyacrylamide gel), followed by 35 S-labeled ponsin-2 blot overlay. (A2) 35 S-Labeled l-afadin blot overlay. Various GST-fused proteins of ponsin-2 (5 pmol each) were subjected to SDS-PAGE (8% polyacrylamide gel), followed by 35 S-labeled l-afadin blot overlay. (B) Affinity chromatography. (B1) Binding region of l-afadin. His6–l-Afadin-C199 or His6–l-afadin-C132 was incubated with GST–ponsin-2-C (the COOH-terminal half) or GST alone immobilized on glutathione-Sepharose beads. Each original sample and eluate were subjected to SDS-PAGE (15% polyacrylamide gel), followed by protein staining with Coomassie brilliant blue. (B2) Binding region of ponsin. His6– l-afadin-C199 was incubated with various GST-fused proteins of ponsin-2 immobilized on glutathione-Sepharose beads. Each eluate was subjected to SDS-PAGE (12% polyacrylamide gel), followed by Western blot analysis using the anti–l-afadin antibody. [PR(1)] the first proline-rich region (aa 1219–1229), [PR(2)] the second proline-rich region (aa 1372–1399), [PR(3)] the third proline-rich region (aa 1691–1713), (C199) His6–l-afadin-C199, (C132) His6–l-afadin-C132, (F) GST–ponsin-2-F (the full length), (N) GST–ponsin-2-N (the NH 2 -terminal half), (C) GST–ponsin-2-C, [SH3(1+2)] GST– ponsin-2-SH3(1+2) (the first and second SH3 domains), [SH3(2+3)] GST–ponsin-2-SH3(2+3) (the second and third SH3 domains), [SH3(1)] GST–ponsin-2-SH3(1) (the first SH3 domain), [SH3(2)] GST–ponsin-2-SH3(2) (the second SH3 domain), and [SH3(3)] GST– ponsin-2-SH3(3) (the third SH3 domain). The results are representative of three independent experiments.

Techniques Used: Binding Assay, Labeling, SDS Page, Affinity Chromatography, Incubation, Staining, Western Blot

25) Product Images from "DCNL1 Functions as a Substrate Sensor and Activator of Cullin 2-RING Ligase"

Article Title: DCNL1 Functions as a Substrate Sensor and Activator of Cullin 2-RING Ligase

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.01342-12

CUL2 is not required for DCNL1 binding to VHL. (A, B, and C) HEK293 cells were transfected with the indicated plasmids. Cells were treated with MG132 for 4 h. Cells were lysed, immunoprecipitated using an anti-FLAG antibody, resolved by SDS-PAGE, and immunoblotted using the indicated antibodies. (D) Bacterially purified GST or GST-DCNL1 was incubated with bacterially purified His-VHL(1–155). His pulldown (PD) was performed using nickel agarose; proteins were then resolved by SDS-PAGE and detected by immunoblotting using antibodies directed against GST and His. (E) Rabbit reticulocyte lysate in vitro translated HA-HIF1α and HA-VHL were incubated with bacterially purified GST or GST-DCNL1. GST pulldown was performed using glutathione-Sepharose. Proteins were resolved by SDS-PAGE and immunoblotted using the indicated antibodies. (F) HA-HIF1α was in vitro translated using either rabbit reticulocyte lysate or wheat germ extract and incubated with bacterially purified GST or GST-DCNL1. GST pulldown was performed using glutathione-Sepharose, and bound proteins were resolved by SDS-PAGE and detected using the indicated antibodies. WCE, whole-cell extract; IP, immunoprecipitation.
Figure Legend Snippet: CUL2 is not required for DCNL1 binding to VHL. (A, B, and C) HEK293 cells were transfected with the indicated plasmids. Cells were treated with MG132 for 4 h. Cells were lysed, immunoprecipitated using an anti-FLAG antibody, resolved by SDS-PAGE, and immunoblotted using the indicated antibodies. (D) Bacterially purified GST or GST-DCNL1 was incubated with bacterially purified His-VHL(1–155). His pulldown (PD) was performed using nickel agarose; proteins were then resolved by SDS-PAGE and detected by immunoblotting using antibodies directed against GST and His. (E) Rabbit reticulocyte lysate in vitro translated HA-HIF1α and HA-VHL were incubated with bacterially purified GST or GST-DCNL1. GST pulldown was performed using glutathione-Sepharose. Proteins were resolved by SDS-PAGE and immunoblotted using the indicated antibodies. (F) HA-HIF1α was in vitro translated using either rabbit reticulocyte lysate or wheat germ extract and incubated with bacterially purified GST or GST-DCNL1. GST pulldown was performed using glutathione-Sepharose, and bound proteins were resolved by SDS-PAGE and detected using the indicated antibodies. WCE, whole-cell extract; IP, immunoprecipitation.

Techniques Used: Binding Assay, Transfection, Immunoprecipitation, SDS Page, Purification, Incubation, In Vitro

HIF1α(555–575) peptide has reduced capacity to promote VHL-DCNL1 interaction. The indicated constructs were in vitro translated in rabbit reticulocyte lysate and incubated with bacterially purified GST or GST-DCNL1. GST pulldown (PD) was performed using glutathione-Sepharose, and bound proteins were resolved by SDS-PAGE and detected using the indicated antibodies.
Figure Legend Snippet: HIF1α(555–575) peptide has reduced capacity to promote VHL-DCNL1 interaction. The indicated constructs were in vitro translated in rabbit reticulocyte lysate and incubated with bacterially purified GST or GST-DCNL1. GST pulldown (PD) was performed using glutathione-Sepharose, and bound proteins were resolved by SDS-PAGE and detected using the indicated antibodies.

Techniques Used: Construct, In Vitro, Incubation, Purification, SDS Page

DCNL1 is recruited through VHL to initiate CUL2 neddylation and HIF1α degradation. (A) Schematic diagram of the indicated proteins and their capacity for recruitment of CUL2 and DCNL1. (B) The indicated constructs were in vitro translated in rabbit reticulocyte lysate and incubated with bacterially purified GST or GST-DCNL1. A GST pulldown (PD) was performed using glutathione-Sepharose, and bound proteins were resolved by SDS-PAGE and detected using the indicated antibodies. (C) HEK293 cells were transfected with the indicated plasmids. Cells were harvested at 48 h posttransfection and immunoprecipitated using an anti-GAL4 antibody. Proteins were separated by SDS-PAGE and immunoblotted using the indicated antibodies. (D) The indicated plasmids were in vitro translated, and in vitro ubiquitylation was performed in the presence (+) or absence (−) of FLAG-ubiquitin (FLAG-Ub) and S100 extracts generated from VHL-null or VHL-reconstituted cells. The reaction mixtures were immunoprecipitated using anti-GAL4 antibody, resolved by SDS-PAGE, and immunoblotted using the indicated antibodies. WCE, whole-cell extract; IP, immunoprecipitation; IB, immunoblot.
Figure Legend Snippet: DCNL1 is recruited through VHL to initiate CUL2 neddylation and HIF1α degradation. (A) Schematic diagram of the indicated proteins and their capacity for recruitment of CUL2 and DCNL1. (B) The indicated constructs were in vitro translated in rabbit reticulocyte lysate and incubated with bacterially purified GST or GST-DCNL1. A GST pulldown (PD) was performed using glutathione-Sepharose, and bound proteins were resolved by SDS-PAGE and detected using the indicated antibodies. (C) HEK293 cells were transfected with the indicated plasmids. Cells were harvested at 48 h posttransfection and immunoprecipitated using an anti-GAL4 antibody. Proteins were separated by SDS-PAGE and immunoblotted using the indicated antibodies. (D) The indicated plasmids were in vitro translated, and in vitro ubiquitylation was performed in the presence (+) or absence (−) of FLAG-ubiquitin (FLAG-Ub) and S100 extracts generated from VHL-null or VHL-reconstituted cells. The reaction mixtures were immunoprecipitated using anti-GAL4 antibody, resolved by SDS-PAGE, and immunoblotted using the indicated antibodies. WCE, whole-cell extract; IP, immunoprecipitation; IB, immunoblot.

Techniques Used: Construct, In Vitro, Incubation, Purification, SDS Page, Transfection, Immunoprecipitation, Generated

26) Product Images from "RAD51 and MRE11 dependent reassembly of uncoupled CMG helicase complex at collapsed replication forks"

Article Title: RAD51 and MRE11 dependent reassembly of uncoupled CMG helicase complex at collapsed replication forks

Journal: Nature structural & molecular biology

doi: 10.1038/nsmb.2177

The role of PCNA in DNA replication and chromatin association of replication proteins upon fork collapse. In ( a ) replication of sperm nuclei incubated in extracts for 80 min in the presence of 1 μg ml −1 aphidicolin and 0, 0.73, 0.37, 0.18 U μl −1 S1 nuclease and PCNA wild type (WT), PCNA K164R (KR), PCNA Y249A Y250A (YA) or PCNA K164R Y249A Y250A (KR YA) recombinant proteins. Replication products were resolved by neutral agarose gel and subjected to autoradiography (left). Signal intensities were quantified and reported in the graph (right). ( b ) Binding to chromatin of the indicated proteins was monitored by immunoblotting of chromatin treated with 200 J m −2 UV or incubated in extracts treated with 1 μg ml −1 aphidicolin, 0.97 U μl −1 S1 nuclease or 0.1 U μl −1 EcoR1 and recombinant PCNA wild type (WT), PCNA K164R (KR) or PCNA Y249A Y250A (YA) as indicated. 0.5 μl egg extract was loaded as a control (Ext). ( c ) The interaction of PCNA and replication proteins in egg extract was monitored by incubation of His-tagged wild type and mutant PCNA proteins followed by pull down with Ni-NTA sepharose. The interacting proteins were detected by immunoblotting as indicated.
Figure Legend Snippet: The role of PCNA in DNA replication and chromatin association of replication proteins upon fork collapse. In ( a ) replication of sperm nuclei incubated in extracts for 80 min in the presence of 1 μg ml −1 aphidicolin and 0, 0.73, 0.37, 0.18 U μl −1 S1 nuclease and PCNA wild type (WT), PCNA K164R (KR), PCNA Y249A Y250A (YA) or PCNA K164R Y249A Y250A (KR YA) recombinant proteins. Replication products were resolved by neutral agarose gel and subjected to autoradiography (left). Signal intensities were quantified and reported in the graph (right). ( b ) Binding to chromatin of the indicated proteins was monitored by immunoblotting of chromatin treated with 200 J m −2 UV or incubated in extracts treated with 1 μg ml −1 aphidicolin, 0.97 U μl −1 S1 nuclease or 0.1 U μl −1 EcoR1 and recombinant PCNA wild type (WT), PCNA K164R (KR) or PCNA Y249A Y250A (YA) as indicated. 0.5 μl egg extract was loaded as a control (Ext). ( c ) The interaction of PCNA and replication proteins in egg extract was monitored by incubation of His-tagged wild type and mutant PCNA proteins followed by pull down with Ni-NTA sepharose. The interacting proteins were detected by immunoblotting as indicated.

Techniques Used: Incubation, Recombinant, Agarose Gel Electrophoresis, Autoradiography, Binding Assay, Mutagenesis

RAD51 is required for origin independent fork restart and reloading of replisome components after fork collapse. In ( a ) and ( b ) replication fork restart was monitored following incubation of sperm nuclei in the 1 st extract for 60 min with or without 10 μg/ml aphidicolin and then transferring nuclear fractions that were untreated or briefly incubated with Mung bean nuclease to a 2 nd extract containing 320 nM geminin, 1 mM roscovitine and GST or GST-BRC4 ( a ), or to mock or RAD51-depleted extracts containing 25, 50, 100 nM recombinant RAD51 ( b ). Replication products were monitored by incorporation of 32 P-dATP added to the 2 nd extract and resolved by alkaline ( a ) or neutral agarose gel ( b ) and subjected to autoradiography. Quantification of signals is shown at the bottom of the gel in ( a ) and in the graph ( b ). In ( c ) chromatin binding of RAD51 and CDC45 was monitored in egg extracts that were mock or RAD51 depleted and supplemented with the indicated amount of recombinant RAD51 (rRAD51). The status of replication fork proteins bound to chromatin isolated from extracts treated as in ( a ) is shown in ( d ).
Figure Legend Snippet: RAD51 is required for origin independent fork restart and reloading of replisome components after fork collapse. In ( a ) and ( b ) replication fork restart was monitored following incubation of sperm nuclei in the 1 st extract for 60 min with or without 10 μg/ml aphidicolin and then transferring nuclear fractions that were untreated or briefly incubated with Mung bean nuclease to a 2 nd extract containing 320 nM geminin, 1 mM roscovitine and GST or GST-BRC4 ( a ), or to mock or RAD51-depleted extracts containing 25, 50, 100 nM recombinant RAD51 ( b ). Replication products were monitored by incorporation of 32 P-dATP added to the 2 nd extract and resolved by alkaline ( a ) or neutral agarose gel ( b ) and subjected to autoradiography. Quantification of signals is shown at the bottom of the gel in ( a ) and in the graph ( b ). In ( c ) chromatin binding of RAD51 and CDC45 was monitored in egg extracts that were mock or RAD51 depleted and supplemented with the indicated amount of recombinant RAD51 (rRAD51). The status of replication fork proteins bound to chromatin isolated from extracts treated as in ( a ) is shown in ( d ).

Techniques Used: Incubation, Transferring, Recombinant, Agarose Gel Electrophoresis, Autoradiography, Binding Assay, Isolation

MRE11 nuclease activity is required for DNA replication upon fork collapse. In ( a ) and ( b ) the effects of MRE11 nuclease inhibitor mirin on replication of sperm nuclei that were untreated or treated with MMS in the presence or absence of GST-BRC4 ( a ) or on sperm nuclei incubated in extracts treated with 0, 0.73, 0.37, 0.18 U μl −1 S1 nuclease and aphidicolin were monitored ( b ). Replication products were monitored by 32 P-dATP incorporation and resolved by neutral agarose gels, which were subjected to autoradiography. Signal intensities were reported in the graphs. In ( c ) the effect of mirin on replication proteins bound to chromatin isolated after 50 min incubation in extracts treated with 0, 1.46, 0.73, 0.37 and 0.18 U μl −1 S1 nuclease was analysed. In ( d ) the binding of the indicated fork proteins to chromatin incubated for 45 min in egg extracts that were untreated or supplemented with 0.73 U μl −1 S1 nuclease and mirin was monitored following protein crosslinking, sonication induced DNA fragmentation and immunoprecipitation with control and anti-CDC45 serum. * non-specific band. Ext: 0.5 μl egg extract was loaded as a control in ( c ) and ( d ).
Figure Legend Snippet: MRE11 nuclease activity is required for DNA replication upon fork collapse. In ( a ) and ( b ) the effects of MRE11 nuclease inhibitor mirin on replication of sperm nuclei that were untreated or treated with MMS in the presence or absence of GST-BRC4 ( a ) or on sperm nuclei incubated in extracts treated with 0, 0.73, 0.37, 0.18 U μl −1 S1 nuclease and aphidicolin were monitored ( b ). Replication products were monitored by 32 P-dATP incorporation and resolved by neutral agarose gels, which were subjected to autoradiography. Signal intensities were reported in the graphs. In ( c ) the effect of mirin on replication proteins bound to chromatin isolated after 50 min incubation in extracts treated with 0, 1.46, 0.73, 0.37 and 0.18 U μl −1 S1 nuclease was analysed. In ( d ) the binding of the indicated fork proteins to chromatin incubated for 45 min in egg extracts that were untreated or supplemented with 0.73 U μl −1 S1 nuclease and mirin was monitored following protein crosslinking, sonication induced DNA fragmentation and immunoprecipitation with control and anti-CDC45 serum. * non-specific band. Ext: 0.5 μl egg extract was loaded as a control in ( c ) and ( d ).

Techniques Used: Activity Assay, Incubation, Autoradiography, Isolation, Binding Assay, Sonication, Immunoprecipitation

27) Product Images from "Scd6 targets eIF4G to repress translation: RGG-motif proteins as a class of eIF4G-binding proteins"

Article Title: Scd6 targets eIF4G to repress translation: RGG-motif proteins as a class of eIF4G-binding proteins

Journal: Molecular cell

doi: 10.1016/j.molcel.2011.11.026

Scd6 forms a tri-complex with eIF4E/G on cap A) Northern analysis of luciferase mRNA containing BoxB repeats pulled down from translation extracts with GST-lambda protein (see procedures) +/− purified His-tagged Scd6 at 6uM concentration. B) Western analysis of proteins coming along with luciferase mRNA containing BoxB using antibodies indicated. C) 7-methyl-GTP sepharose pull downs were performed using recombinant purified eIF4G/4E and Scd6 proteins (see procedures). D) 7-methyl-GTP sepharose pull downs performed from yeast translation extracts in presence of Scd6 or Scd6ΔRGG. E) FLAG-pull downs from translation reactions to pull down recombinant Scd6. 20 ul translation reactions were diluted to 200ul followed by addition of FLAG-agarose beads. Binding was performed for an hour with end to end rotation followed by three washes of beads for 10′ each. The beads were then boiled in SDS-loading buffer and analyzed by SDS-PAGE followed by western blotting. Typically 10% of total reaction was loaded in the ‘input’ lanes and 100% of total pulled-down material was loaded in ‘pellet’ lanes. Bands in C and D were quantified by using Image J program.
Figure Legend Snippet: Scd6 forms a tri-complex with eIF4E/G on cap A) Northern analysis of luciferase mRNA containing BoxB repeats pulled down from translation extracts with GST-lambda protein (see procedures) +/− purified His-tagged Scd6 at 6uM concentration. B) Western analysis of proteins coming along with luciferase mRNA containing BoxB using antibodies indicated. C) 7-methyl-GTP sepharose pull downs were performed using recombinant purified eIF4G/4E and Scd6 proteins (see procedures). D) 7-methyl-GTP sepharose pull downs performed from yeast translation extracts in presence of Scd6 or Scd6ΔRGG. E) FLAG-pull downs from translation reactions to pull down recombinant Scd6. 20 ul translation reactions were diluted to 200ul followed by addition of FLAG-agarose beads. Binding was performed for an hour with end to end rotation followed by three washes of beads for 10′ each. The beads were then boiled in SDS-loading buffer and analyzed by SDS-PAGE followed by western blotting. Typically 10% of total reaction was loaded in the ‘input’ lanes and 100% of total pulled-down material was loaded in ‘pellet’ lanes. Bands in C and D were quantified by using Image J program.

Techniques Used: Northern Blot, Luciferase, Purification, Concentration Assay, Western Blot, Recombinant, Binding Assay, SDS Page

Scd6 does not destabilize Pab1-4G or 4A-4G interactions A) 7-methyl-GTP sepharose pull downs were performed to look at interaction of GST-eIF4G/eIF4E complex with recombinant Pab1 in presence of Scd6 or Scd6ΔRGG protein as described in materials and methods. 8ug of GST-eIF4G/eIF4E was incubated with wt or mutant Scd6 protein for 1h at 4°C with end-to-end rotation. Following this Pab1 was added to reaction mixture and incubated for 1h with end-to-end rotation. Finally, 7-methyl-GTP sepharose was added to reaction mix and incubated for 2h. Washing was performed as described in materials and methods. eIF4G and Pab1 were detected with polyclonal antibodies. B) Glutathione pull down assay to analyze effect of purified Scd6 on eIF4A-eIF4E/G interaction. Incubation and washing conditions were same as mentioned in A. eIF4G and eIF4A were detected with polyclonal antibodies. The anti-eIF4A antibody cross-reacts with purified Scd6 (contains both His and FLAG-tag). The middle panel depicts ponceau-stained blot with clearly visible wt and mutant Scd6 proteins. This was done since eIF4A and Scd6ΔRGG run at similar position (in top panel). Typically 10% of total reaction was loaded in the ‘input’ lanes and 50 or 100% of total pulled-down material was loaded in ‘pellet’ lanes.
Figure Legend Snippet: Scd6 does not destabilize Pab1-4G or 4A-4G interactions A) 7-methyl-GTP sepharose pull downs were performed to look at interaction of GST-eIF4G/eIF4E complex with recombinant Pab1 in presence of Scd6 or Scd6ΔRGG protein as described in materials and methods. 8ug of GST-eIF4G/eIF4E was incubated with wt or mutant Scd6 protein for 1h at 4°C with end-to-end rotation. Following this Pab1 was added to reaction mixture and incubated for 1h with end-to-end rotation. Finally, 7-methyl-GTP sepharose was added to reaction mix and incubated for 2h. Washing was performed as described in materials and methods. eIF4G and Pab1 were detected with polyclonal antibodies. B) Glutathione pull down assay to analyze effect of purified Scd6 on eIF4A-eIF4E/G interaction. Incubation and washing conditions were same as mentioned in A. eIF4G and eIF4A were detected with polyclonal antibodies. The anti-eIF4A antibody cross-reacts with purified Scd6 (contains both His and FLAG-tag). The middle panel depicts ponceau-stained blot with clearly visible wt and mutant Scd6 proteins. This was done since eIF4A and Scd6ΔRGG run at similar position (in top panel). Typically 10% of total reaction was loaded in the ‘input’ lanes and 50 or 100% of total pulled-down material was loaded in ‘pellet’ lanes.

Techniques Used: Recombinant, Incubation, Mutagenesis, Pull Down Assay, Purification, FLAG-tag, Staining

Scd6 binds eIF4G ). D) Glutathione sepharose pull downs performed to look at interaction of purified GST-4G/E with recombinant Scd6 and Scd6ΔRGG mutant protein. Scd6 protein was detected with anti-FLAG antibody following manufacturer’s instructions. E) Glutathione sepharose pull downs were performed to look at interaction of GST-4G in bacterial extracts with recombinant Scd6 and Scd6ΔRGG mutant protein. 6ug of each protein was used in 200ul reaction mixture. Scd6 protein was detected with anti-FLAG antibody following manufacturer’s instructions. Typically 10% of total reaction was loaded in the ‘input’ lanes and 100% of total pulled-down material was loaded in ‘pellet’ lanes.
Figure Legend Snippet: Scd6 binds eIF4G ). D) Glutathione sepharose pull downs performed to look at interaction of purified GST-4G/E with recombinant Scd6 and Scd6ΔRGG mutant protein. Scd6 protein was detected with anti-FLAG antibody following manufacturer’s instructions. E) Glutathione sepharose pull downs were performed to look at interaction of GST-4G in bacterial extracts with recombinant Scd6 and Scd6ΔRGG mutant protein. 6ug of each protein was used in 200ul reaction mixture. Scd6 protein was detected with anti-FLAG antibody following manufacturer’s instructions. Typically 10% of total reaction was loaded in the ‘input’ lanes and 100% of total pulled-down material was loaded in ‘pellet’ lanes.

Techniques Used: Purification, Recombinant, Mutagenesis

28) Product Images from "Cbl-b Positively Regulates Btk-mediated Activation of Phospholipase C-?2 in B Cells"

Article Title: Cbl-b Positively Regulates Btk-mediated Activation of Phospholipase C-?2 in B Cells

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20020068

Characterization of the PLC-γ2/Ca 2+ signaling, Rap1 activation, and MAPKs activation in Cbl-b–deficient DT40 cells. (A) Ca 2+ mobilization in wild-type DT40 cells (red) and Cbl-b–deficient cells, and clones C3-1 (blue) and C3-3 (green). [Ca 2+ ] i were monitored by spectrophotometer after stimulation with 2 μg/ml M4 or 0.5 μg/ml ionomycin. Ca 2+ release from intracellular Ca 2+ store was monitored after stimulation with 2 μg/ml M4 in the presence of 1 mM EGTA (+EGTA). Ca 2+ was restored to the media to evaluate extracellular Ca 2+ influx. Arrows indicate the time point of the addition of M4 (α − μ), ionomycin, and CaCl 2 . Similar results were obtained using two other Cbl-b–deficient clones. (B) IP 3 production in wild-type and Cbl-b–deficient DT40 cells. 2 × 10 6 cells were stimulated with 2 μg M4 for the indicated time period and IP 3 production was measured. Results are the mean ± SEM of three independent experiments. (C) Tyrosine phosphorylation of PLC-γ2 in wild-type and Cbl-b–deficient DT40 cells. 10 7 cells were lysed in 1% NP-40 lysis buffer at the indicated time points after stimulation of 4 μg/ml M4. Anti–PLC-γ2 immunoprecipitates from the lysates were subjected to Western blot analysis with anti-phosphotyrosine mAb (top) and anti–PLC-γ2 Ab (bottom). (D) Rap1 activation in wild-type and Cbl-b–deficient DT40 cells. 1.25 × 10 7 cells were lysed in 1% NP-40 lysis buffer at the indicated time points after stimulation of 5 μg/ml M4. Cell lysates were precipitated with GST-RalGDS(RBD) immobilized on glutathione Sepharose, and bound proteins were analyzed by anti-Rap1 immunoblotting. (E–G) BCR-induced ERK2, p38, and JNK activation in wild-type and Cbl-b–deficient DT40 cells. 2.5 × 10 7 cells were lysed at the indicated time points after stimulation of 4 μg/ml M4. (E) Anti-ERK2, (F) anti-p38, or (G) anti-JNK1 immunoprecipitates from the lysates were subjected to kinase reaction using GST-Elk, GST-ATF2, or GST–c-Jun as an exogenous substrate, respectively. The protein levels in the immunoprecipitates are shown in E–G. All experiments were performed more than three times.
Figure Legend Snippet: Characterization of the PLC-γ2/Ca 2+ signaling, Rap1 activation, and MAPKs activation in Cbl-b–deficient DT40 cells. (A) Ca 2+ mobilization in wild-type DT40 cells (red) and Cbl-b–deficient cells, and clones C3-1 (blue) and C3-3 (green). [Ca 2+ ] i were monitored by spectrophotometer after stimulation with 2 μg/ml M4 or 0.5 μg/ml ionomycin. Ca 2+ release from intracellular Ca 2+ store was monitored after stimulation with 2 μg/ml M4 in the presence of 1 mM EGTA (+EGTA). Ca 2+ was restored to the media to evaluate extracellular Ca 2+ influx. Arrows indicate the time point of the addition of M4 (α − μ), ionomycin, and CaCl 2 . Similar results were obtained using two other Cbl-b–deficient clones. (B) IP 3 production in wild-type and Cbl-b–deficient DT40 cells. 2 × 10 6 cells were stimulated with 2 μg M4 for the indicated time period and IP 3 production was measured. Results are the mean ± SEM of three independent experiments. (C) Tyrosine phosphorylation of PLC-γ2 in wild-type and Cbl-b–deficient DT40 cells. 10 7 cells were lysed in 1% NP-40 lysis buffer at the indicated time points after stimulation of 4 μg/ml M4. Anti–PLC-γ2 immunoprecipitates from the lysates were subjected to Western blot analysis with anti-phosphotyrosine mAb (top) and anti–PLC-γ2 Ab (bottom). (D) Rap1 activation in wild-type and Cbl-b–deficient DT40 cells. 1.25 × 10 7 cells were lysed in 1% NP-40 lysis buffer at the indicated time points after stimulation of 5 μg/ml M4. Cell lysates were precipitated with GST-RalGDS(RBD) immobilized on glutathione Sepharose, and bound proteins were analyzed by anti-Rap1 immunoblotting. (E–G) BCR-induced ERK2, p38, and JNK activation in wild-type and Cbl-b–deficient DT40 cells. 2.5 × 10 7 cells were lysed at the indicated time points after stimulation of 4 μg/ml M4. (E) Anti-ERK2, (F) anti-p38, or (G) anti-JNK1 immunoprecipitates from the lysates were subjected to kinase reaction using GST-Elk, GST-ATF2, or GST–c-Jun as an exogenous substrate, respectively. The protein levels in the immunoprecipitates are shown in E–G. All experiments were performed more than three times.

Techniques Used: Planar Chromatography, Activation Assay, Clone Assay, Spectrophotometry, Lysis, Western Blot

Tyrosine phosphorylation of Cbl-b and generation of Cbl-b–deficient DT40 B cells. (A) At the indicated time points after stimulation of 8 μg/ml M4, wild-type, Lyn-, Syk-, or Btk-deficient DT40 cells (2.5 × 10 7 ) were lysed in 1% NP-40 lysis buffer containing 10 mg/ml digitonin. Anti–Cbl-b (C-20) immunoprecipitates from the lysates were subjected to Western blot analysis with anti-phosphotyrosine mAb (top) and anti–Cbl-b (C-20) Ab (bottom). (B) Structure of the chicken cbl-b allele, the targeting vector, and the mutated allele. Restriction sites for EcoRI (E) are indicated. (C) Southern blot analysis of wild-type and targeted DT40 cells. EcoRI-digested genomic DNAs were separated on an agarose gel, blotted, and hybridized with the chicken cbl-b cDNA probe (B, 5′ probe). (D) Northern blot analysis of wild-type and Cbl-b–deficient DT40 cells using chicken cDNA probe for cbl-b (top) or β-actin (bottom). (E) Protein expression of Cbl-b in wild-type and Cbl-b–deficient DT40 cells. Immunoprecipitates with anti–Cbl-b (C-20) Ab were prepared from wild-type and Cbl-b–deficient DT40 cells and subjected to Western blot analysis using anti–Cbl-b (C-20) Ab. (F) BCR expression on the surface of wild-type (wt), Cbl-b–deficient ( cbl-b − , C3-1, and C3-3), and various DT40 derivatives were monitored by flow cytometry. Unstained cells were used as the negative controls (dashed histogram). Wild-type and Cbl-b–deficient cells expressing T7-tagged Btk are indicated as T7-Btk/wt and T7-Btk/ cbl-b − , respectively. Cbl-b–deficient cells (C3-3) expressing wild-type Cbl-b, G298E mutant Cbl-b, COOH-terminal deletion mutant Cbl-b (1-444 amino acids), and C373A mutant Cbl-b are shown as WT/ cbl-b − , GE/ cbl-b − , N/ cbl-b − , and CA/ cbl-b − , respectively. The x and y axes for the histograms indicate fluorescence intensity (four-decade log scales) and relative cell number, respectively.
Figure Legend Snippet: Tyrosine phosphorylation of Cbl-b and generation of Cbl-b–deficient DT40 B cells. (A) At the indicated time points after stimulation of 8 μg/ml M4, wild-type, Lyn-, Syk-, or Btk-deficient DT40 cells (2.5 × 10 7 ) were lysed in 1% NP-40 lysis buffer containing 10 mg/ml digitonin. Anti–Cbl-b (C-20) immunoprecipitates from the lysates were subjected to Western blot analysis with anti-phosphotyrosine mAb (top) and anti–Cbl-b (C-20) Ab (bottom). (B) Structure of the chicken cbl-b allele, the targeting vector, and the mutated allele. Restriction sites for EcoRI (E) are indicated. (C) Southern blot analysis of wild-type and targeted DT40 cells. EcoRI-digested genomic DNAs were separated on an agarose gel, blotted, and hybridized with the chicken cbl-b cDNA probe (B, 5′ probe). (D) Northern blot analysis of wild-type and Cbl-b–deficient DT40 cells using chicken cDNA probe for cbl-b (top) or β-actin (bottom). (E) Protein expression of Cbl-b in wild-type and Cbl-b–deficient DT40 cells. Immunoprecipitates with anti–Cbl-b (C-20) Ab were prepared from wild-type and Cbl-b–deficient DT40 cells and subjected to Western blot analysis using anti–Cbl-b (C-20) Ab. (F) BCR expression on the surface of wild-type (wt), Cbl-b–deficient ( cbl-b − , C3-1, and C3-3), and various DT40 derivatives were monitored by flow cytometry. Unstained cells were used as the negative controls (dashed histogram). Wild-type and Cbl-b–deficient cells expressing T7-tagged Btk are indicated as T7-Btk/wt and T7-Btk/ cbl-b − , respectively. Cbl-b–deficient cells (C3-3) expressing wild-type Cbl-b, G298E mutant Cbl-b, COOH-terminal deletion mutant Cbl-b (1-444 amino acids), and C373A mutant Cbl-b are shown as WT/ cbl-b − , GE/ cbl-b − , N/ cbl-b − , and CA/ cbl-b − , respectively. The x and y axes for the histograms indicate fluorescence intensity (four-decade log scales) and relative cell number, respectively.

Techniques Used: Lysis, Western Blot, Plasmid Preparation, Southern Blot, Agarose Gel Electrophoresis, Northern Blot, Expressing, Flow Cytometry, Cytometry, Mutagenesis, Fluorescence

Effects of mutations in Cbl-b on BCR-induced Ca 2+ mobilization. (A) Schematic representation of the wild-type and mutant Cbl-b proteins. 4H, four-helix bundle; EF, EF hand; SH2, Src homology 2; RF, RING finger; PRO, proline-rich region; LZ, leucine zipper. (B) Expression and tyrosine phosphorylation of wild-type or mutant Cbl-b in Cbl-b–deficient cells. The indicated cells were lysed in 1% NP-40 lysis buffer at 0, 1, and 3 min after stimulation of 4 μg/ml M4. Anti–Cbl-b (G-1) immunoprecipitates from the lysates were subjected to Western blot analysis with anti-phosphotyrosine mAb (top) and anti–Cbl-b (G-1) mAb (bottom). The positions of WT, GE, N, and CA Cbl-b are indicated. (C) BCR-induced Ca 2+ mobilization in wild-type cells, Cbl-b–deficient cells (C3-3), and Cbl-b–deficient cells expressing wild-type Cbl-b (WT/ cbl-b − ), G298E Cbl-b (GE/ cbl-b − ), a COOH-terminal deletion mutant (N/ cbl-b − ), or C373A Cbl-b (CA/ cbl-b − ). [Ca 2+ ] i were monitored by spectrophotometer after stimulation with 2 μg/ml M4 (−EGTA). Ca 2+ release from intracellular Ca 2+ store was monitored after stimulation of 2 μg/ml M4 in the presence of 1 mM EGTA (+EGTA), and Ca 2+ was restored to the media to evaluate extracellular Ca 2+ influx. Arrows indicate the time point of the addition of M4 (α − μ) and CaCl 2 . The representative results from four independent clones of each cell type are shown. (D) Association of Cbl-b with PLC-γ2. The indicated cells were lysed in 1% NP-40 lysis buffer at 0, 1, and 3 min after stimulation of 4 μg/ml M4. Anti–PLC-γ2 immunoprecipitates from the lysates were subjected to Western blot analysis with anti–Cbl-b (G-1) mAb (top) and anti–PLC-γ2 Ab (bottom). The positions of WT, GE, N, and CA Cbl-b are indicated. (E) In vitro binding of Cbl-b with the SH3 domains of Btk and PLC-γ2. WEHI-231 cells overexpressing Cbl-b (C85-11) and unstimulated (−) or stimulated (+) with anti-IgM (10 μg/10 7 cells) for 3 min, were lysed in 1% NP-40 lysis buffer, and the lysates were incubated with the indicated GST fusion proteins immobilized on glutathione Sepharose. The bound proteins or lysates (1/10 input) were separated by SDS-PAGE and immunoblotted with anti–Cbl-b (G-1) mAb (left). Expression of the GST fusion proteins was confirmed by Coomassie staining (right). (C–E) Experiments were performed more than three times.
Figure Legend Snippet: Effects of mutations in Cbl-b on BCR-induced Ca 2+ mobilization. (A) Schematic representation of the wild-type and mutant Cbl-b proteins. 4H, four-helix bundle; EF, EF hand; SH2, Src homology 2; RF, RING finger; PRO, proline-rich region; LZ, leucine zipper. (B) Expression and tyrosine phosphorylation of wild-type or mutant Cbl-b in Cbl-b–deficient cells. The indicated cells were lysed in 1% NP-40 lysis buffer at 0, 1, and 3 min after stimulation of 4 μg/ml M4. Anti–Cbl-b (G-1) immunoprecipitates from the lysates were subjected to Western blot analysis with anti-phosphotyrosine mAb (top) and anti–Cbl-b (G-1) mAb (bottom). The positions of WT, GE, N, and CA Cbl-b are indicated. (C) BCR-induced Ca 2+ mobilization in wild-type cells, Cbl-b–deficient cells (C3-3), and Cbl-b–deficient cells expressing wild-type Cbl-b (WT/ cbl-b − ), G298E Cbl-b (GE/ cbl-b − ), a COOH-terminal deletion mutant (N/ cbl-b − ), or C373A Cbl-b (CA/ cbl-b − ). [Ca 2+ ] i were monitored by spectrophotometer after stimulation with 2 μg/ml M4 (−EGTA). Ca 2+ release from intracellular Ca 2+ store was monitored after stimulation of 2 μg/ml M4 in the presence of 1 mM EGTA (+EGTA), and Ca 2+ was restored to the media to evaluate extracellular Ca 2+ influx. Arrows indicate the time point of the addition of M4 (α − μ) and CaCl 2 . The representative results from four independent clones of each cell type are shown. (D) Association of Cbl-b with PLC-γ2. The indicated cells were lysed in 1% NP-40 lysis buffer at 0, 1, and 3 min after stimulation of 4 μg/ml M4. Anti–PLC-γ2 immunoprecipitates from the lysates were subjected to Western blot analysis with anti–Cbl-b (G-1) mAb (top) and anti–PLC-γ2 Ab (bottom). The positions of WT, GE, N, and CA Cbl-b are indicated. (E) In vitro binding of Cbl-b with the SH3 domains of Btk and PLC-γ2. WEHI-231 cells overexpressing Cbl-b (C85-11) and unstimulated (−) or stimulated (+) with anti-IgM (10 μg/10 7 cells) for 3 min, were lysed in 1% NP-40 lysis buffer, and the lysates were incubated with the indicated GST fusion proteins immobilized on glutathione Sepharose. The bound proteins or lysates (1/10 input) were separated by SDS-PAGE and immunoblotted with anti–Cbl-b (G-1) mAb (left). Expression of the GST fusion proteins was confirmed by Coomassie staining (right). (C–E) Experiments were performed more than three times.

Techniques Used: Mutagenesis, Expressing, Lysis, Western Blot, Spectrophotometry, Clone Assay, Planar Chromatography, In Vitro, Binding Assay, Incubation, SDS Page, Staining

29) Product Images from "Localized suppression of RhoA activity by Tyr31/118-phosphorylated paxillin in cell adhesion and migration"

Article Title: Localized suppression of RhoA activity by Tyr31/118-phosphorylated paxillin in cell adhesion and migration

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200202117

Protein binding to specific paxillin peptides. 500 μg of TGFβ-treated NMuMG cell lysate was incubated with 250 pmol of the phosphorylated (P) and unphosphorylated (−) forms of paxillin peptides immobilized on streptavidin-Sepharose beads, in a total volume of 500 μl for 1 h at 4°C. Proteins precipitated were analyzed by immunoblotting using antibodies as indicated. The numbers indicate the peptides corresponding to the tyrosine phosphorylation sites of paxillin. 10 μg of total cell lysate was included as controls (Total).
Figure Legend Snippet: Protein binding to specific paxillin peptides. 500 μg of TGFβ-treated NMuMG cell lysate was incubated with 250 pmol of the phosphorylated (P) and unphosphorylated (−) forms of paxillin peptides immobilized on streptavidin-Sepharose beads, in a total volume of 500 μl for 1 h at 4°C. Proteins precipitated were analyzed by immunoblotting using antibodies as indicated. The numbers indicate the peptides corresponding to the tyrosine phosphorylation sites of paxillin. 10 μg of total cell lysate was included as controls (Total).

Techniques Used: Protein Binding, Incubation

Competition of Tyr31/118-phosphorylated paxillin with p190RhoGAP for binding to p120RasGAP. (A) 3Y1/v-Src cell lysates were incubated with the p120RasGAP GST-SH2-SH3-SH2 protein, preincubated with a paxillin peptide (or combinations of peptides) as in Fig. 3 E. The control included the GST fusion protein without incubation with peptides (−). Proteins precipitated were analyzed by anti-p190RhoGAP immunoblotting. (B and C) COS7 cells were transiently transfected with 0, 1, or 3 μg of pEGFP-RhoGAP, as indicated (B), or transfected with 3 μg of pBabe/EGFP-paxillin (WT) or 3 μg of pBabe/EGFP-paxillin 2X (2X), as indicate (C). Mock-transfected cells were also included (C, P). 24 h later, cells were detached from culture dishes by incubation with PBS containing 5 mM EDTA, washed, and replated onto collagen type I–coated dishes in the presence of 0.5% BSA for 1 h. Cells were then lysed, and protein coprecipitation was analyzed using an anti-paxillin antibody coupled with anti–mouse IgG-Sepharose beads (B), or an anti-RasGAP antibody (clone B4F8) coupled with protein A-Sepharose beads (C). Protein immunoblotting was performed as indicated, with immunoprecipitated proteins (IP) and total cell lysates (Total). (D) Amounts of p190RhoGAP coprecipitating with p120RasGAP using antibody B4F8 were analyzed, as above, with cell lysates from parental NMuMG cells (P), or NMuMG cells expressing EGFP-paxillin (WT) or cells expressing the 2X mutant (2X), all pretreated with TGFβ. (C and D) To show the difference in the amounts of p190RhoGAP coprecipitating with p120RasGAP, a longer exposure of the immunoblots shown on the top of each panel, is also shown at the bottom. Exposure time was 15 s for the top, and 2 min for the bottom. (B–D) Total cell lysates were included as controls (Total). Molecular sizes are shown on the left. ▴, endogenous paxillin; ▵, EGFP-tagged paxillin.
Figure Legend Snippet: Competition of Tyr31/118-phosphorylated paxillin with p190RhoGAP for binding to p120RasGAP. (A) 3Y1/v-Src cell lysates were incubated with the p120RasGAP GST-SH2-SH3-SH2 protein, preincubated with a paxillin peptide (or combinations of peptides) as in Fig. 3 E. The control included the GST fusion protein without incubation with peptides (−). Proteins precipitated were analyzed by anti-p190RhoGAP immunoblotting. (B and C) COS7 cells were transiently transfected with 0, 1, or 3 μg of pEGFP-RhoGAP, as indicated (B), or transfected with 3 μg of pBabe/EGFP-paxillin (WT) or 3 μg of pBabe/EGFP-paxillin 2X (2X), as indicate (C). Mock-transfected cells were also included (C, P). 24 h later, cells were detached from culture dishes by incubation with PBS containing 5 mM EDTA, washed, and replated onto collagen type I–coated dishes in the presence of 0.5% BSA for 1 h. Cells were then lysed, and protein coprecipitation was analyzed using an anti-paxillin antibody coupled with anti–mouse IgG-Sepharose beads (B), or an anti-RasGAP antibody (clone B4F8) coupled with protein A-Sepharose beads (C). Protein immunoblotting was performed as indicated, with immunoprecipitated proteins (IP) and total cell lysates (Total). (D) Amounts of p190RhoGAP coprecipitating with p120RasGAP using antibody B4F8 were analyzed, as above, with cell lysates from parental NMuMG cells (P), or NMuMG cells expressing EGFP-paxillin (WT) or cells expressing the 2X mutant (2X), all pretreated with TGFβ. (C and D) To show the difference in the amounts of p190RhoGAP coprecipitating with p120RasGAP, a longer exposure of the immunoblots shown on the top of each panel, is also shown at the bottom. Exposure time was 15 s for the top, and 2 min for the bottom. (B–D) Total cell lysates were included as controls (Total). Molecular sizes are shown on the left. ▴, endogenous paxillin; ▵, EGFP-tagged paxillin.

Techniques Used: Binding Assay, Incubation, Transfection, Immunoprecipitation, Expressing, Mutagenesis, Western Blot

Tyr31/118-phosphorylated paxillin binds to p120RasGAP, via the two SH2 domains. (A) TGFβ-treated (+) or untreated (−) NMuMG cell lysates were immunoprecipitated with a mouse monoclonal anti-paxillin antibody coupled with anti–mouse IgG-Sepharose beads, and subjected to immunoblotting, as indicated. (B and C) TGFβ-treated (+) or untreated (−) NMuMG cell lysates were incubated with GST fusion proteins, each corresponding to various parts of the SH2-SH3-SH2 domain of p120RasGAP (B), and proteins bound to the beads were analyzed by immunoblotting, as indicated (C). Asterisks indicate mutations in the SH2 domains. Coomassie blue staining of the GST proteins used are also shown (CBB). (D) The GST-SH2-SH3-SH2 protein was preincubated with 100 μM each of the phosphorylated or unphosphorylated paxillin peptides (or combinations of peptides), as indicated, before being incubated with TGFβ-treated NMuMG cell lysate, and bound proteins were subjected to anti-paxillin blotting. (E) TGFβ-treated NMuMG cell lysates were incubated with a mouse monoclonal anti-paxillin antibody (IP) or the GST-SH2-SH3-SH2 protein, and bound proteins were analyzed using phosphorylation site-specific antibodies for paxillin ( pY31 , pY40 , pY118 and pY181) or an anti-paxillin antibody, as indicated. 20 pmol each of the paxillin peptides used in Fig. 2 was spotted onto filters and used as controls (right). Total cell lysate (Total) or cell lysate incubated with GST were included as controls, and the molecular sizes in kD are shown, where necessary.
Figure Legend Snippet: Tyr31/118-phosphorylated paxillin binds to p120RasGAP, via the two SH2 domains. (A) TGFβ-treated (+) or untreated (−) NMuMG cell lysates were immunoprecipitated with a mouse monoclonal anti-paxillin antibody coupled with anti–mouse IgG-Sepharose beads, and subjected to immunoblotting, as indicated. (B and C) TGFβ-treated (+) or untreated (−) NMuMG cell lysates were incubated with GST fusion proteins, each corresponding to various parts of the SH2-SH3-SH2 domain of p120RasGAP (B), and proteins bound to the beads were analyzed by immunoblotting, as indicated (C). Asterisks indicate mutations in the SH2 domains. Coomassie blue staining of the GST proteins used are also shown (CBB). (D) The GST-SH2-SH3-SH2 protein was preincubated with 100 μM each of the phosphorylated or unphosphorylated paxillin peptides (or combinations of peptides), as indicated, before being incubated with TGFβ-treated NMuMG cell lysate, and bound proteins were subjected to anti-paxillin blotting. (E) TGFβ-treated NMuMG cell lysates were incubated with a mouse monoclonal anti-paxillin antibody (IP) or the GST-SH2-SH3-SH2 protein, and bound proteins were analyzed using phosphorylation site-specific antibodies for paxillin ( pY31 , pY40 , pY118 and pY181) or an anti-paxillin antibody, as indicated. 20 pmol each of the paxillin peptides used in Fig. 2 was spotted onto filters and used as controls (right). Total cell lysate (Total) or cell lysate incubated with GST were included as controls, and the molecular sizes in kD are shown, where necessary.

Techniques Used: Immunoprecipitation, Incubation, Staining

30) Product Images from "Activity of a Bacterial Cell Envelope Stress Response Is Controlled by the Interaction of a Protein Binding Domain with Different Partners *"

Article Title: Activity of a Bacterial Cell Envelope Stress Response Is Controlled by the Interaction of a Protein Binding Domain with Different Partners *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.614107

Evidence that interaction of the PspC C-terminal domain with PspA or PspB is mutually exclusive in vitro . A , summary of the protocol used for the GST fusion protein two-phase membrane lysate pulldown assay. B , immunoblot analysis. In Experiment A , GST or GST fused to the PspC C-terminal domain ( GST-PspC CT ) was bound to glutathione-Sepharose (beads) and incubated with a detergent-solubilized membrane lysate from a Y. enterocolitica strain in which the only core Psp protein present was PspA ( prey lysate 1 ). After washing, proteins were recovered from half of the beads by boiling in SDS-PAGE sample buffer ( Elution 1 ). The other half of the beads was incubated with a second detergent-solubilized membrane lysate from a Y. enterocolitica strain in which the only core Psp protein present was PspB ( prey lysate 2 ). After washing, proteins were recovered by boiling in SDS-PAGE sample buffer ( Elution 2 ). Experiment B was done similarly, except that the order of incubation with the PspA and PspB membrane lysates was reversed. For each experiment, membrane lysates ( Inputs ) and recovered proteins ( Elutions ) were analyzed by SDS-PAGE and immunoblotting with PspA or PspB antiserum. The GST fusion protein in each elution was detected by Ponceau S staining of the immunoblot membrane (for experiments A and B the elution samples for Ponceau S staining were run on the same gels, but irrelevant lanes between elutions 1 and 2 have been removed).
Figure Legend Snippet: Evidence that interaction of the PspC C-terminal domain with PspA or PspB is mutually exclusive in vitro . A , summary of the protocol used for the GST fusion protein two-phase membrane lysate pulldown assay. B , immunoblot analysis. In Experiment A , GST or GST fused to the PspC C-terminal domain ( GST-PspC CT ) was bound to glutathione-Sepharose (beads) and incubated with a detergent-solubilized membrane lysate from a Y. enterocolitica strain in which the only core Psp protein present was PspA ( prey lysate 1 ). After washing, proteins were recovered from half of the beads by boiling in SDS-PAGE sample buffer ( Elution 1 ). The other half of the beads was incubated with a second detergent-solubilized membrane lysate from a Y. enterocolitica strain in which the only core Psp protein present was PspB ( prey lysate 2 ). After washing, proteins were recovered by boiling in SDS-PAGE sample buffer ( Elution 2 ). Experiment B was done similarly, except that the order of incubation with the PspA and PspB membrane lysates was reversed. For each experiment, membrane lysates ( Inputs ) and recovered proteins ( Elutions ) were analyzed by SDS-PAGE and immunoblotting with PspA or PspB antiserum. The GST fusion protein in each elution was detected by Ponceau S staining of the immunoblot membrane (for experiments A and B the elution samples for Ponceau S staining were run on the same gels, but irrelevant lanes between elutions 1 and 2 have been removed).

Techniques Used: In Vitro, Incubation, SDS Page, Staining

GST-PspC CT fusion protein pulldown assay. GST, GST fused to the PspC C-terminal domain ( GST-PspC CT ), or a derivative with the V125D mutation ( GST-PspC CT-V125D ) was bound to glutathione-Sepharose (beads) and incubated with a detergent-solubilized membrane lysate from a Y. enterocolitica strain with all core Psp proteins (Psp + ) or in which the only core Psp proteins present were PspA or PspB as indicated ( Prey ). After washing, proteins were recovered by boiling in SDS-PAGE sample buffer. Membrane lysates ( Inputs ) and recovered proteins ( Elutions ) were analyzed by SDS-PAGE and immunoblotting with PspA or PspB antiserum. The GST fusion protein in each elution was detected by Ponceau S staining of the immunoblot membrane.
Figure Legend Snippet: GST-PspC CT fusion protein pulldown assay. GST, GST fused to the PspC C-terminal domain ( GST-PspC CT ), or a derivative with the V125D mutation ( GST-PspC CT-V125D ) was bound to glutathione-Sepharose (beads) and incubated with a detergent-solubilized membrane lysate from a Y. enterocolitica strain with all core Psp proteins (Psp + ) or in which the only core Psp proteins present were PspA or PspB as indicated ( Prey ). After washing, proteins were recovered by boiling in SDS-PAGE sample buffer. Membrane lysates ( Inputs ) and recovered proteins ( Elutions ) were analyzed by SDS-PAGE and immunoblotting with PspA or PspB antiserum. The GST fusion protein in each elution was detected by Ponceau S staining of the immunoblot membrane.

Techniques Used: Mutagenesis, Incubation, SDS Page, Staining

In vitro GST/MBP fusion protein interaction assay. GST, GST fused to the PspC C-terminal domain ( GST-PspC CT ), or a derivative with the V125D mutation ( GST-PspC CT-V125D ) were bound to glutathione-Sepharose (beads) and incubated with 15 μg of MBP fused to the C-terminal domain of PspB ( MBP-PspB CT ) or to LacZα ( MBP-LacZ α). After washing, proteins were recovered by boiling in SDS-PAGE sample buffer. Samples of each purified MBP-fusion protein ( Inputs ) and the recovered proteins ( Elutions ) were analyzed by SDS-PAGE and immunoblotting with anti-MBP or anti-GST monoclonal antibodies. MBP-LacZα underwent apparent degradation during purification, leading to the isolation of both full-length and truncated protein.
Figure Legend Snippet: In vitro GST/MBP fusion protein interaction assay. GST, GST fused to the PspC C-terminal domain ( GST-PspC CT ), or a derivative with the V125D mutation ( GST-PspC CT-V125D ) were bound to glutathione-Sepharose (beads) and incubated with 15 μg of MBP fused to the C-terminal domain of PspB ( MBP-PspB CT ) or to LacZα ( MBP-LacZ α). After washing, proteins were recovered by boiling in SDS-PAGE sample buffer. Samples of each purified MBP-fusion protein ( Inputs ) and the recovered proteins ( Elutions ) were analyzed by SDS-PAGE and immunoblotting with anti-MBP or anti-GST monoclonal antibodies. MBP-LacZα underwent apparent degradation during purification, leading to the isolation of both full-length and truncated protein.

Techniques Used: In Vitro, Protein Interaction Assay, Mutagenesis, Incubation, SDS Page, Purification, Isolation

31) Product Images from "Metastasis-Associated Protein 1 Interacts with NRIF3, an Estrogen-Inducible Nuclear Receptor Coregulator"

Article Title: Metastasis-Associated Protein 1 Interacts with NRIF3, an Estrogen-Inducible Nuclear Receptor Coregulator

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.24.15.6581-6591.2004

Modulation of NRIF3 mRNA expression by estrogen and antiestrogen. (A and B) MCF-7 cells were treated with E2 (10 −9 M) in the absence or presence of ICI or 4-hydroxytamoxifen (10 −8 M) for the indicated times. Total RNA (20 μg) was analyzed by Northern blotting with an NRIF3 cDNA probe. Glyceraldehhyde-3-phosphate dehydrogenase (GAPDH) and total RNA agarose gel picture were used as loading controls. (C) Estrogen induces NRIF3 transcription. MCF-7 cells were treated with cycloheximide (50 μg/ml) or actinomycin D (10 μg/ml) in the presence or absence of E2 (10 −9 M) for 3 h. Total RNA was isolated, and the levels of NRIF3 mRNA were detected by Northern blotting. (D) E2 signaling promotes NRIF3 interaction with the endogenous ER pathway. Schematic presentation of the tentative NRIF3 promoter (upper panel). MCF-7 cells were treated for 3 h with or without estrogen (10 −9 M). Subsequently, the cells were processed for the chromatin immunoprecipitation assay with anti-ER-α antibody, and PCR was performed with the primers from the NRIF3 promoter. The bottom panel shows PCR analysis of the input DNA. The middle panel demonstrates PCR analysis of the NRIF3 promoter fragments associated with ER-α. The other two pair of primers did not work.
Figure Legend Snippet: Modulation of NRIF3 mRNA expression by estrogen and antiestrogen. (A and B) MCF-7 cells were treated with E2 (10 −9 M) in the absence or presence of ICI or 4-hydroxytamoxifen (10 −8 M) for the indicated times. Total RNA (20 μg) was analyzed by Northern blotting with an NRIF3 cDNA probe. Glyceraldehhyde-3-phosphate dehydrogenase (GAPDH) and total RNA agarose gel picture were used as loading controls. (C) Estrogen induces NRIF3 transcription. MCF-7 cells were treated with cycloheximide (50 μg/ml) or actinomycin D (10 μg/ml) in the presence or absence of E2 (10 −9 M) for 3 h. Total RNA was isolated, and the levels of NRIF3 mRNA were detected by Northern blotting. (D) E2 signaling promotes NRIF3 interaction with the endogenous ER pathway. Schematic presentation of the tentative NRIF3 promoter (upper panel). MCF-7 cells were treated for 3 h with or without estrogen (10 −9 M). Subsequently, the cells were processed for the chromatin immunoprecipitation assay with anti-ER-α antibody, and PCR was performed with the primers from the NRIF3 promoter. The bottom panel shows PCR analysis of the input DNA. The middle panel demonstrates PCR analysis of the NRIF3 promoter fragments associated with ER-α. The other two pair of primers did not work.

Techniques Used: Expressing, Northern Blot, Agarose Gel Electrophoresis, Isolation, Chromatin Immunoprecipitation, Polymerase Chain Reaction

NRIF3 stimulates the expression of E2-responsive genes and associates with the ERE-responsive chromatin. (A) NRIF3 stable cell lines. Upper panels show expression of T7-NRIF3 protein by Western blotting with T7 monoclonal antibody. Vinculin expression was used as a loading control. Lower panels show Northern blot analysis of NRIF3 mRNA expression. Glyceraldehhyde-3-phosphate dehydrogenase (GAPDH) and total RNA agarose gel picture were used as loading controls. (B) Equal amounts of cDNA probes from pcDNA and NRIF3 clone 19 were generated by reverse transcription-PCR, hybridized with two identical human estrogen signaling pathway GE array blots, and detected by phosphorimager scanning. The signals of the two blots were normalized to β-actin. Relative expression of E2-responsive genes in the NRIF3 clone was compared with that of the pcDNA clone. (C) Total RNA was made from MCF-7 stable pooled clones expressing pcDNA and NRIF3, hybridized with pS2 cDNA (upper panel), and reprobed with the HMG1 cDNA probe (middle panel). The same blot was hybridized with the glyceraldehhyde-3-phosphate dehydrogenase cDNA probe (bottom panel). (D) E2 induces while tamoxifen inhibits the expression of PS2 and HMG1 mRNA expression. (E) Recruitment of NRIF3 to estrogen target gene promoter. Stable clones expressing either T7-NRIF3 or control vector in MCF-7 cells were treated with estrogen (10 −9 M) or with ICI (10 −7 M) or both for the indicated times. Cells were cross-linked and processed for the chromatin immunoprecipitation assay by immunoprecipitation with anti-T7 antibody, and PCR was performed on the pS2 promoter.
Figure Legend Snippet: NRIF3 stimulates the expression of E2-responsive genes and associates with the ERE-responsive chromatin. (A) NRIF3 stable cell lines. Upper panels show expression of T7-NRIF3 protein by Western blotting with T7 monoclonal antibody. Vinculin expression was used as a loading control. Lower panels show Northern blot analysis of NRIF3 mRNA expression. Glyceraldehhyde-3-phosphate dehydrogenase (GAPDH) and total RNA agarose gel picture were used as loading controls. (B) Equal amounts of cDNA probes from pcDNA and NRIF3 clone 19 were generated by reverse transcription-PCR, hybridized with two identical human estrogen signaling pathway GE array blots, and detected by phosphorimager scanning. The signals of the two blots were normalized to β-actin. Relative expression of E2-responsive genes in the NRIF3 clone was compared with that of the pcDNA clone. (C) Total RNA was made from MCF-7 stable pooled clones expressing pcDNA and NRIF3, hybridized with pS2 cDNA (upper panel), and reprobed with the HMG1 cDNA probe (middle panel). The same blot was hybridized with the glyceraldehhyde-3-phosphate dehydrogenase cDNA probe (bottom panel). (D) E2 induces while tamoxifen inhibits the expression of PS2 and HMG1 mRNA expression. (E) Recruitment of NRIF3 to estrogen target gene promoter. Stable clones expressing either T7-NRIF3 or control vector in MCF-7 cells were treated with estrogen (10 −9 M) or with ICI (10 −7 M) or both for the indicated times. Cells were cross-linked and processed for the chromatin immunoprecipitation assay by immunoprecipitation with anti-T7 antibody, and PCR was performed on the pS2 promoter.

Techniques Used: Expressing, Stable Transfection, Western Blot, Northern Blot, Agarose Gel Electrophoresis, Generated, Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Chromatin Immunoprecipitation, Immunoprecipitation

Interference with NRIF3 expression reduces ER-responsive pathways. (A) Interference with NRIF3 expression decrease the expression of PS2 and HMG1. MCF-7 cells were treated with control siRNA or NRIF3 siRNA for 72 h, total RNA was isolated, and expression of NRIF3, PS2, and HMG1 mRNAs was determined by Northern hybridization. β-Actin and total RNA agarose gel picture were used as loading controls. (B) Downregulation of endogenous NRIF3 severely inhibits the ability of estrogen to induce ER-inducible genes. MCF-7 cells grown in the presence of 3% DCC serum were treated with control siRNA or NRIF3 siRNA for 72 h, total RNA was isolated, and expression of NRIF3, PS2, and HMG1 mRNAs was determined by Northern hybridization. (C) Effect of endogenous NRIF3 on ER transactivation. ZR-75 cells were transfected with control or NRIF3 siRNA. After 24 h, cells were transfected with ERE-luciferase (200 ng/well) on six-well plates in the absence or presence of NRIF3 (150 ng/well) and treated with or without estrogen for 15 h, and ERE-luciferase activity was measured ( n = 3). (D) NRIF3 induces activation function domain 2 (AF2)-driven transactivation. HeLa cells were transfected with control or NRIF3 siRNA. After 24 h, cells were transfected with GAL4-luciferase (200 ng/well) and GAL4-AF2 (200 ng/well) in the absence or presence of NRIF3 (50 ng/well) and treated with or without estrogen for 15 h, and luciferase activity was measured ( n = 3).
Figure Legend Snippet: Interference with NRIF3 expression reduces ER-responsive pathways. (A) Interference with NRIF3 expression decrease the expression of PS2 and HMG1. MCF-7 cells were treated with control siRNA or NRIF3 siRNA for 72 h, total RNA was isolated, and expression of NRIF3, PS2, and HMG1 mRNAs was determined by Northern hybridization. β-Actin and total RNA agarose gel picture were used as loading controls. (B) Downregulation of endogenous NRIF3 severely inhibits the ability of estrogen to induce ER-inducible genes. MCF-7 cells grown in the presence of 3% DCC serum were treated with control siRNA or NRIF3 siRNA for 72 h, total RNA was isolated, and expression of NRIF3, PS2, and HMG1 mRNAs was determined by Northern hybridization. (C) Effect of endogenous NRIF3 on ER transactivation. ZR-75 cells were transfected with control or NRIF3 siRNA. After 24 h, cells were transfected with ERE-luciferase (200 ng/well) on six-well plates in the absence or presence of NRIF3 (150 ng/well) and treated with or without estrogen for 15 h, and ERE-luciferase activity was measured ( n = 3). (D) NRIF3 induces activation function domain 2 (AF2)-driven transactivation. HeLa cells were transfected with control or NRIF3 siRNA. After 24 h, cells were transfected with GAL4-luciferase (200 ng/well) and GAL4-AF2 (200 ng/well) in the absence or presence of NRIF3 (50 ng/well) and treated with or without estrogen for 15 h, and luciferase activity was measured ( n = 3).

Techniques Used: Expressing, Isolation, Northern Blot, Hybridization, Agarose Gel Electrophoresis, Droplet Countercurrent Chromatography, Transfection, Luciferase, Activity Assay, Activation Assay

32) Product Images from "Molecular Networks in FGF Signaling: Flotillin-1 and Cbl-Associated Protein Compete for the Binding to Fibroblast Growth Factor Receptor Substrate 2"

Article Title: Molecular Networks in FGF Signaling: Flotillin-1 and Cbl-Associated Protein Compete for the Binding to Fibroblast Growth Factor Receptor Substrate 2

Journal: PLoS ONE

doi: 10.1371/journal.pone.0029739

FRS2 directly interacts with Cbl-associated protein. (A) Yeast two-hybrid analysis of the interaction between FRS2 and CAP domains. (B) Structure of the CAP-GST constructs used. (C) and (D) Interaction of purified FRS2-His and CAP-GST proteins. CAP-GST fusion proteins were immobilized on sepharose and tested for the binding of purified FRS2-His. Upper blot shows the bound FRS2-His (anti-His antibody), lower blot the ponceau staining of the GST proteins. 1 µg of FRS2-His was used as a positive control. (E) Quantification of the binding of FRS2 to various CAP domains. A binding of FRS2 significantly higher than background was seen with the full-length CAP, delta-SoHo and the third SH3 domain. (F) Endogenous FRS2 was immunoprecipitated from Hep3B cells, and the binding of endogenous CAP was tested. Please note that several isoforms of CAP are present in Hep3B cells, of which only one appears to bind FRS2.
Figure Legend Snippet: FRS2 directly interacts with Cbl-associated protein. (A) Yeast two-hybrid analysis of the interaction between FRS2 and CAP domains. (B) Structure of the CAP-GST constructs used. (C) and (D) Interaction of purified FRS2-His and CAP-GST proteins. CAP-GST fusion proteins were immobilized on sepharose and tested for the binding of purified FRS2-His. Upper blot shows the bound FRS2-His (anti-His antibody), lower blot the ponceau staining of the GST proteins. 1 µg of FRS2-His was used as a positive control. (E) Quantification of the binding of FRS2 to various CAP domains. A binding of FRS2 significantly higher than background was seen with the full-length CAP, delta-SoHo and the third SH3 domain. (F) Endogenous FRS2 was immunoprecipitated from Hep3B cells, and the binding of endogenous CAP was tested. Please note that several isoforms of CAP are present in Hep3B cells, of which only one appears to bind FRS2.

Techniques Used: Construct, Purification, Binding Assay, Staining, Positive Control, Immunoprecipitation

Flot-1 and CAP compete for the binding to FRS2. CAP-GST was immobilized to sepharose and incubated with HeLa cell lysates in the presence of increasing amounts (1–5 µg) of purified FRS2-His. The binding of endogenous flot-1 from the lysates was analyzed by Western blot (upper blot). Middle panel shows the blot for FRS2-His and the lowermost one a ponceau staining of the GST proteins.
Figure Legend Snippet: Flot-1 and CAP compete for the binding to FRS2. CAP-GST was immobilized to sepharose and incubated with HeLa cell lysates in the presence of increasing amounts (1–5 µg) of purified FRS2-His. The binding of endogenous flot-1 from the lysates was analyzed by Western blot (upper blot). Middle panel shows the blot for FRS2-His and the lowermost one a ponceau staining of the GST proteins.

Techniques Used: Binding Assay, Incubation, Purification, Western Blot, Staining

Overexpression of FRS2 does not compensate for the signaling defects in flot-1 knockdown cells. FGF receptor and flot-1 compete for the binding to FRS2. (A) Flot-1 was knocked down in HeLa cells by means of siRNAs and the cells were transfected with FRS2-CFP. Starved cells were stimulated with FGF for 5 min, and the activation of Akt (uppermost blot) and ERK2 (3 rd blot) was measured with phospho-specific antibodies. The third blot from the bottom shows the analysis of the transfection efficiency of FRS2-CFP and of the 2 nd one the knockdown efficiency of flot-1. Lowermost blot (GAPDH) shows equal protein loading. (B) Purified FRS2-GST was immobilized on sepharose and incubated with lysates of HeLa cells transfected with increasing amounts of FGFR-myc (0.5 to 2 µg). The binding of endogenous flot-1 from these lysates was tested (upper blot). (C) Quantification of the flot-1 bound to FRS2. In the presence of increasing amounts of FGFR, the binding is significantly reduced. (D) Expression of FGFR was verified by Western blot.
Figure Legend Snippet: Overexpression of FRS2 does not compensate for the signaling defects in flot-1 knockdown cells. FGF receptor and flot-1 compete for the binding to FRS2. (A) Flot-1 was knocked down in HeLa cells by means of siRNAs and the cells were transfected with FRS2-CFP. Starved cells were stimulated with FGF for 5 min, and the activation of Akt (uppermost blot) and ERK2 (3 rd blot) was measured with phospho-specific antibodies. The third blot from the bottom shows the analysis of the transfection efficiency of FRS2-CFP and of the 2 nd one the knockdown efficiency of flot-1. Lowermost blot (GAPDH) shows equal protein loading. (B) Purified FRS2-GST was immobilized on sepharose and incubated with lysates of HeLa cells transfected with increasing amounts of FGFR-myc (0.5 to 2 µg). The binding of endogenous flot-1 from these lysates was tested (upper blot). (C) Quantification of the flot-1 bound to FRS2. In the presence of increasing amounts of FGFR, the binding is significantly reduced. (D) Expression of FGFR was verified by Western blot.

Techniques Used: Over Expression, Binding Assay, Transfection, Activation Assay, Purification, Incubation, Expressing, Western Blot

33) Product Images from "Inhibition of Nuclear Import by the Proapoptotic Protein CC3"

Article Title: Inhibition of Nuclear Import by the Proapoptotic Protein CC3

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.24.16.7091-7101.2004

CC3 associates with transportin and the NPC in vitro and in vivo. (A) In vitro-translated transportin polypeptides: full-length (TRN), N-terminal amino acids 1 to 581 (TRN-N), and C-terminal amino acids 581 to 890 (TRN-C) were incubated with GST or GST-CC3. Complexes were recovered with glutathione-Sepharose, washed, resolved by SDS-PAGE, and detected by autoradiography. (B) HeLa cell extracts were subjected to immunoprecipitation with a control immunoglobulin G (IgG) or anti-transportin antibody. The immune complexes were resolved by electrophoresis and analyzed by Western blotting with antibodies to transportin (TRN) and CC3. (C) Purified CC3 at 1 μM was mixed with GST or GST-transportin for 1 h at 4°C. Complexes were recovered with glutathione-Sepharose, resolved by SDS-PAGE, and detected by Western blotting with anti-CC3 antibody. (D) HeLa cells were processed for immunofluorescence with the anti-CC3 rabbit polyclonal antibody and anti-NPC mouse monoclonal MAb414 (Covance), followed by detection with anti-rabbit IgG-CY3 (red) and anti-mouse IgG-FITC (green), and analyzed by confocal microscopy. (E) MAb414 was used to immunoprecipitate components of the NPC from HeLa cells extracts as described in Materials and Methods. The immune complexes were analyzed by Western blotting with MAb414 and antibodies to CC3.
Figure Legend Snippet: CC3 associates with transportin and the NPC in vitro and in vivo. (A) In vitro-translated transportin polypeptides: full-length (TRN), N-terminal amino acids 1 to 581 (TRN-N), and C-terminal amino acids 581 to 890 (TRN-C) were incubated with GST or GST-CC3. Complexes were recovered with glutathione-Sepharose, washed, resolved by SDS-PAGE, and detected by autoradiography. (B) HeLa cell extracts were subjected to immunoprecipitation with a control immunoglobulin G (IgG) or anti-transportin antibody. The immune complexes were resolved by electrophoresis and analyzed by Western blotting with antibodies to transportin (TRN) and CC3. (C) Purified CC3 at 1 μM was mixed with GST or GST-transportin for 1 h at 4°C. Complexes were recovered with glutathione-Sepharose, resolved by SDS-PAGE, and detected by Western blotting with anti-CC3 antibody. (D) HeLa cells were processed for immunofluorescence with the anti-CC3 rabbit polyclonal antibody and anti-NPC mouse monoclonal MAb414 (Covance), followed by detection with anti-rabbit IgG-CY3 (red) and anti-mouse IgG-FITC (green), and analyzed by confocal microscopy. (E) MAb414 was used to immunoprecipitate components of the NPC from HeLa cells extracts as described in Materials and Methods. The immune complexes were analyzed by Western blotting with MAb414 and antibodies to CC3.

Techniques Used: In Vitro, In Vivo, Incubation, SDS Page, Autoradiography, Immunoprecipitation, Electrophoresis, Western Blot, Purification, Immunofluorescence, Confocal Microscopy

Identification of proteins associated with CC3. HeLa cells were metabolically labeled with [ 35 S]methionine and lysed. Cytosolic protein extracts were incubated with GST or GST-CC3. Bound proteins were recovered on glutathione-Sepharose, resolved by SDS-PAGE, and detected by autoradiography.
Figure Legend Snippet: Identification of proteins associated with CC3. HeLa cells were metabolically labeled with [ 35 S]methionine and lysed. Cytosolic protein extracts were incubated with GST or GST-CC3. Bound proteins were recovered on glutathione-Sepharose, resolved by SDS-PAGE, and detected by autoradiography.

Techniques Used: Metabolic Labelling, Labeling, Incubation, SDS Page, Autoradiography

Interactions of CC3 with NPC, transportin, and exportin 4 are not sensitive to RanGTP. (A) FITC-labeled GST-M9 or GST-CC3 at 0.5 μM were added to permeabilized HeLa cells in transport buffer with or without 50 μg of HeLa cytosolic extract and 8 μM RanQ69L. After incubation for 20 min at room temperature, the cells were fixed and examined by fluorescence microscopy. (B) In vitro-translated transportin (lane 1) was incubated with GST-CC3 in the absence (lane 2) or in the presence of 1, 4, or 8 μM RanQ69L (lanes 3 to 5). Complexes were recovered with glutathione-Sepharose, resolved by SDS-PAGE, and detected by autoradiography. (C) His-exportin 4 at 5 μM was incubated with GST (lane 1) or GST-CC3 without RanQ69L (lane 2) or in the presence of 2.5, 5, or 10 μM RanQ69L (lanes 3 to 5) for 1 h at 4°C. Complexes were recovered with glutathione-Sepharose, resolved by SDS-PAGE, and detected by staining with Coomassie blue. (D) In vitro-translated transportin was incubated with His-RanQ69L in the absence (lane 3) or presence of 1, 4, and 8 μM GST-CC3 (lanes 4 to 6). Complexes were recovered with Ni-NTA agarose and analyzed by SDS-PAGE, followed by autoradiography. Lane 2 shows the amount of transportin that was incubated without RanQ69L and bound to the Ni-NTA agarose in a nonspecific manner. (E) In vitro-translated transportin was incubated with GST-M9 in absence (lane 2) or in presence of 1, 4, or 8 μM CC3 (lanes 3 to 5). Complexes were analyzed as described for panel B.
Figure Legend Snippet: Interactions of CC3 with NPC, transportin, and exportin 4 are not sensitive to RanGTP. (A) FITC-labeled GST-M9 or GST-CC3 at 0.5 μM were added to permeabilized HeLa cells in transport buffer with or without 50 μg of HeLa cytosolic extract and 8 μM RanQ69L. After incubation for 20 min at room temperature, the cells were fixed and examined by fluorescence microscopy. (B) In vitro-translated transportin (lane 1) was incubated with GST-CC3 in the absence (lane 2) or in the presence of 1, 4, or 8 μM RanQ69L (lanes 3 to 5). Complexes were recovered with glutathione-Sepharose, resolved by SDS-PAGE, and detected by autoradiography. (C) His-exportin 4 at 5 μM was incubated with GST (lane 1) or GST-CC3 without RanQ69L (lane 2) or in the presence of 2.5, 5, or 10 μM RanQ69L (lanes 3 to 5) for 1 h at 4°C. Complexes were recovered with glutathione-Sepharose, resolved by SDS-PAGE, and detected by staining with Coomassie blue. (D) In vitro-translated transportin was incubated with His-RanQ69L in the absence (lane 3) or presence of 1, 4, and 8 μM GST-CC3 (lanes 4 to 6). Complexes were recovered with Ni-NTA agarose and analyzed by SDS-PAGE, followed by autoradiography. Lane 2 shows the amount of transportin that was incubated without RanQ69L and bound to the Ni-NTA agarose in a nonspecific manner. (E) In vitro-translated transportin was incubated with GST-M9 in absence (lane 2) or in presence of 1, 4, or 8 μM CC3 (lanes 3 to 5). Complexes were analyzed as described for panel B.

Techniques Used: Labeling, Incubation, Fluorescence, Microscopy, In Vitro, SDS Page, Autoradiography, Staining

34) Product Images from "PC4 Coactivates MyoD by Relieving the Histone Deacetylase 4-Mediated Inhibition of Myocyte Enhancer Factor 2C"

Article Title: PC4 Coactivates MyoD by Relieving the Histone Deacetylase 4-Mediated Inhibition of Myocyte Enhancer Factor 2C

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.25.6.2242-2259.2005

PC4 interacts in vitro with MEF2C. (A) Binding of in vitro-translated 35 S-labeled MEF2A, MEF2C, and MEF2D to GST-MyoD and GST-PC4. (B) Binding of in vitro-translated 35 ). (D and E) Binding to GST-MEF2C of PC4 mutants with deletions at the carboxyl terminus (D) or at the amino terminus (E). (A, B, D, and E) The indicated [ 35 S]methionine-labeled products in vitro translated in rabbit reticulocyte lysates were incubated with the different GST-fused proteins bound to glutathione-Sepharose 4B. Bound proteins were eluted and analyzed by SDS-8% PAGE, followed by autoradiography. The labeled input products loaded were about 30% (A and B) or 10% (D to E) of the amount used in the pull-down incubations.
Figure Legend Snippet: PC4 interacts in vitro with MEF2C. (A) Binding of in vitro-translated 35 S-labeled MEF2A, MEF2C, and MEF2D to GST-MyoD and GST-PC4. (B) Binding of in vitro-translated 35 ). (D and E) Binding to GST-MEF2C of PC4 mutants with deletions at the carboxyl terminus (D) or at the amino terminus (E). (A, B, D, and E) The indicated [ 35 S]methionine-labeled products in vitro translated in rabbit reticulocyte lysates were incubated with the different GST-fused proteins bound to glutathione-Sepharose 4B. Bound proteins were eluted and analyzed by SDS-8% PAGE, followed by autoradiography. The labeled input products loaded were about 30% (A and B) or 10% (D to E) of the amount used in the pull-down incubations.

Techniques Used: In Vitro, Binding Assay, Labeling, Incubation, Polyacrylamide Gel Electrophoresis, Autoradiography

). (B) C2C12 cells (clone S4 constitutively overexpressing PC4) were transfected with pcDNA-Myc-HDAC4 (or with the empty vector) as indicated, grown in GM or DM for 48 h after transfection, and then lysed and immunoprecipitated with the anti-PC4 antibody A451 covalently bound to Sepharose resin. The immunoprecipitated complexes (IP: a-PC4 panel), as well as the input cell lysates, were analyzed by Western blotting with anti-Myc or anti-PC4 antibodies. (C) C2C7 cells, cultured in GM or DM for 48 h, were lysed and immunoprecipitated with the anti-PC4 antibody covalently bound to Sepharose resin. Immunoprecipitated complexes (IP: a-PC4 panel) and the input cell lysates were analyzed by Western blotting with anti-HDAC4 or anti-PC4 antibodies. As a control (mock IP panel), the immunoprecipitates obtained by using Sepharose resin coupled to preimmune serum were analyzed by Western blotting. For each experiment shown in panels A to C, 1.5 mg of protein lysate was immunoprecipitated and fractionated by SDS-PAGE. Western blot analysis of input lysates was carried out on 1/30 of the lysates used for immunoprecipitation.
Figure Legend Snippet: ). (B) C2C12 cells (clone S4 constitutively overexpressing PC4) were transfected with pcDNA-Myc-HDAC4 (or with the empty vector) as indicated, grown in GM or DM for 48 h after transfection, and then lysed and immunoprecipitated with the anti-PC4 antibody A451 covalently bound to Sepharose resin. The immunoprecipitated complexes (IP: a-PC4 panel), as well as the input cell lysates, were analyzed by Western blotting with anti-Myc or anti-PC4 antibodies. (C) C2C7 cells, cultured in GM or DM for 48 h, were lysed and immunoprecipitated with the anti-PC4 antibody covalently bound to Sepharose resin. Immunoprecipitated complexes (IP: a-PC4 panel) and the input cell lysates were analyzed by Western blotting with anti-HDAC4 or anti-PC4 antibodies. As a control (mock IP panel), the immunoprecipitates obtained by using Sepharose resin coupled to preimmune serum were analyzed by Western blotting. For each experiment shown in panels A to C, 1.5 mg of protein lysate was immunoprecipitated and fractionated by SDS-PAGE. Western blot analysis of input lysates was carried out on 1/30 of the lysates used for immunoprecipitation.

Techniques Used: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Cell Culture, SDS Page

Physical and functional interaction between HDAC4 and PC4. (A) The carboxyl-terminal region of HDAC4 binds PC4. GST pull-down assays were performed by incubating equal amounts of in vitro-translated [ 35 S]methionine-labeled HDAC4 proteins, full-length or truncated mutants, with equimolar amounts of either GST-PC4 or GST proteins bound to glutathione-Sepharose 4B resin. Bound proteins were eluted, separated by SDS-8% PAGE, and analyzed by using a phosphorimager. (B) PC4 does not influence the enzymatic activity of HDAC4. HEK293 cells, grown in 90-mm dishes, were transfected with 12 μg of either pcDNA-Myc-HDAC4 or the empty vector. The precleared lysates were incubated with GST or GST-PC4 and then immunoprecipitated with anti-Myc antibody. As a control, a synthetic peptide corresponding to the Myc epitope was also added in molar excess to samples before immunoprecipitation. Immunoprecipitated proteins, treated or not with trichostatin A (200 nM), were then incubated with preacetylated histones. Deacetylase activities are shown as percent values relative to the level of control samples (transfected with HDAC4 and without GST protein added) set to 100 (the absolute mean dpm values were about 1,400). The bars are the average ± the SEM of three independent experiments performed in duplicate, normalized to the amount of immunoprecipitated proteins by Western blotting (a representative blot is shown in the lower panel). (C) PC4 counteracts the inhibitory effect exerted by the HDAC4 amino-terminal region. The MCK LUC reporter was cotransfected in C3H10T1/2 cells with pEMSV-MyoD (50 ng) and the indicated combinations of HA-pSCT-PC4 (1 μg) and pcDNA-Myc-HDAC4 and pcDNA-Myc-HDAC4 1-611 (5 ng). At 24 h after transfection, cells were placed in DM for 48 h before luciferase assay determination. Luciferase activity was measured as units per microgram of protein normalized to the β-Gal activity. The average fold activity ± the SEM shown (relative to control samples transfected with empty vectors) was calculated from three independent experiments performed in duplicate. ✽, P
Figure Legend Snippet: Physical and functional interaction between HDAC4 and PC4. (A) The carboxyl-terminal region of HDAC4 binds PC4. GST pull-down assays were performed by incubating equal amounts of in vitro-translated [ 35 S]methionine-labeled HDAC4 proteins, full-length or truncated mutants, with equimolar amounts of either GST-PC4 or GST proteins bound to glutathione-Sepharose 4B resin. Bound proteins were eluted, separated by SDS-8% PAGE, and analyzed by using a phosphorimager. (B) PC4 does not influence the enzymatic activity of HDAC4. HEK293 cells, grown in 90-mm dishes, were transfected with 12 μg of either pcDNA-Myc-HDAC4 or the empty vector. The precleared lysates were incubated with GST or GST-PC4 and then immunoprecipitated with anti-Myc antibody. As a control, a synthetic peptide corresponding to the Myc epitope was also added in molar excess to samples before immunoprecipitation. Immunoprecipitated proteins, treated or not with trichostatin A (200 nM), were then incubated with preacetylated histones. Deacetylase activities are shown as percent values relative to the level of control samples (transfected with HDAC4 and without GST protein added) set to 100 (the absolute mean dpm values were about 1,400). The bars are the average ± the SEM of three independent experiments performed in duplicate, normalized to the amount of immunoprecipitated proteins by Western blotting (a representative blot is shown in the lower panel). (C) PC4 counteracts the inhibitory effect exerted by the HDAC4 amino-terminal region. The MCK LUC reporter was cotransfected in C3H10T1/2 cells with pEMSV-MyoD (50 ng) and the indicated combinations of HA-pSCT-PC4 (1 μg) and pcDNA-Myc-HDAC4 and pcDNA-Myc-HDAC4 1-611 (5 ng). At 24 h after transfection, cells were placed in DM for 48 h before luciferase assay determination. Luciferase activity was measured as units per microgram of protein normalized to the β-Gal activity. The average fold activity ± the SEM shown (relative to control samples transfected with empty vectors) was calculated from three independent experiments performed in duplicate. ✽, P

Techniques Used: Functional Assay, In Vitro, Labeling, Polyacrylamide Gel Electrophoresis, Activity Assay, Transfection, Plasmid Preparation, Incubation, Immunoprecipitation, Histone Deacetylase Assay, Western Blot, Luciferase

35) Product Images from "Parkin-Dependent Degradation of the F-Box Protein Fbw7? Promotes Neuronal Survival in Response to Oxidative Stress by Stabilizing Mcl-1"

Article Title: Parkin-Dependent Degradation of the F-Box Protein Fbw7? Promotes Neuronal Survival in Response to Oxidative Stress by Stabilizing Mcl-1

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00535-13

Parkin associates with Fbw7β in primary neurons. (A) Coprecipitation of exogenous parkin with endogenous Fbw7β in primary neurons. Parkin −/− primary embryonic mouse brain neurons were transduced with either a control adenovirus or an adenovirus expressing N-terminally myc-tagged parkin. Lysates were then immunoprecipitated with a mouse monoclonal antibody specific for parkin or equivalent amounts of mouse IgG. After SDS-PAGE and Western blotting, membranes were probed with Fbw7- and parkin-specific antibodies, respectively. (B) Parkin can form complexes with all Fbw7 isoforms. Retrovirus-transduced HEK293A cell lines stably expressing each of the three Fbw7 isoforms as GST fusions were transfected with a plasmid expressing myc-parkin. Fbw7 complexes were precipitated using glutathione (GSH)-Sepharose beads and analyzed by SDS-PAGE and Western blotting using anti-GST or anti-myc tag antibody. (C) Colocalization of parkin and Fbw7β in primary neurons. GFP-parkin and Fbw7β-DsRed were expressed in primary embryonic mouse brain neurons by lentiviral transduction. Proteins were then detected by laser scanning confocal microscopy. Each of the two panels shows the following: upper left, DNA (DAPI); upper right, GFP-parkin; lower left, Fbw7β-DsRed; lower right, merged image. Scale bar, 5 μm. α, anti; IB, immunoblotting.
Figure Legend Snippet: Parkin associates with Fbw7β in primary neurons. (A) Coprecipitation of exogenous parkin with endogenous Fbw7β in primary neurons. Parkin −/− primary embryonic mouse brain neurons were transduced with either a control adenovirus or an adenovirus expressing N-terminally myc-tagged parkin. Lysates were then immunoprecipitated with a mouse monoclonal antibody specific for parkin or equivalent amounts of mouse IgG. After SDS-PAGE and Western blotting, membranes were probed with Fbw7- and parkin-specific antibodies, respectively. (B) Parkin can form complexes with all Fbw7 isoforms. Retrovirus-transduced HEK293A cell lines stably expressing each of the three Fbw7 isoforms as GST fusions were transfected with a plasmid expressing myc-parkin. Fbw7 complexes were precipitated using glutathione (GSH)-Sepharose beads and analyzed by SDS-PAGE and Western blotting using anti-GST or anti-myc tag antibody. (C) Colocalization of parkin and Fbw7β in primary neurons. GFP-parkin and Fbw7β-DsRed were expressed in primary embryonic mouse brain neurons by lentiviral transduction. Proteins were then detected by laser scanning confocal microscopy. Each of the two panels shows the following: upper left, DNA (DAPI); upper right, GFP-parkin; lower left, Fbw7β-DsRed; lower right, merged image. Scale bar, 5 μm. α, anti; IB, immunoblotting.

Techniques Used: Transduction, Expressing, Immunoprecipitation, SDS Page, Western Blot, Stable Transfection, Transfection, Plasmid Preparation, Confocal Microscopy

Parkin ubiquitylates Fbw7. (A) Parkin stimulates ubiquitylation of Fbw7 in neurons. Primary embryonic mouse brain neurons were transduced with adenoviruses expressing either parkin, HA-ubiquitin, or both. Prior to preparation of extracts, neurons were treated with proteasome inhibitors MG132 and PR11. Extracts were immunoprecipitated with either anti-Fbw7 antibody or nonimmune rabbit IgG. Immune complexes were then analyzed by SDS-PAGE and Western blotting using anti-HA antibody (upper panel). The blot was then stripped and developed using anti-Fbw7 antibody (lower panel). The asterisk indicates nonspecific species recognized by anti-Fbw7 antibody. (B) In vitro reconstitution of ubiquitylation of Fbw7 by parkin. Reaction mixtures containing the indicated purified proteins were incubated with a cocktail containing the ubiquitin-activating enzyme (E1), the ubiquitin-conjugating enzyme (E2) UbcH7, and an ATP-regenerating system. GST-ubiquitin conjugates were then purified using glutathione-Sepharose beads and analyzed by SDS-PAGE and Western blotting using anti-Fbw7 antibody. Note that unmodified MBP-Fbw7 is copurified most likely because Fbw7 forms dimers via a dimerization domain amino terminal to its F box, allowing nonubiquitylated Fbw7 to copurifiy with ubiquitylated species. (C) Parkin forms lysine 48 (K48)-linked chains on Fbw7. MBP-Fbw7 from reactions analogous to those described in panel D was purified using amylose-Sepharose beads and analyzed by SDS-PAGE and Western blotting. Parallel samples were analyzed using antiubiquitin antibody or anti-K48-linked ubiquitin antibody. α, anti; Ub, ubiquitin; IP, immunoprecipitation; IB, immunoblotting. The superscript “n” indicates a variable number of ubiquitin molecules conjugated to Fbw7.
Figure Legend Snippet: Parkin ubiquitylates Fbw7. (A) Parkin stimulates ubiquitylation of Fbw7 in neurons. Primary embryonic mouse brain neurons were transduced with adenoviruses expressing either parkin, HA-ubiquitin, or both. Prior to preparation of extracts, neurons were treated with proteasome inhibitors MG132 and PR11. Extracts were immunoprecipitated with either anti-Fbw7 antibody or nonimmune rabbit IgG. Immune complexes were then analyzed by SDS-PAGE and Western blotting using anti-HA antibody (upper panel). The blot was then stripped and developed using anti-Fbw7 antibody (lower panel). The asterisk indicates nonspecific species recognized by anti-Fbw7 antibody. (B) In vitro reconstitution of ubiquitylation of Fbw7 by parkin. Reaction mixtures containing the indicated purified proteins were incubated with a cocktail containing the ubiquitin-activating enzyme (E1), the ubiquitin-conjugating enzyme (E2) UbcH7, and an ATP-regenerating system. GST-ubiquitin conjugates were then purified using glutathione-Sepharose beads and analyzed by SDS-PAGE and Western blotting using anti-Fbw7 antibody. Note that unmodified MBP-Fbw7 is copurified most likely because Fbw7 forms dimers via a dimerization domain amino terminal to its F box, allowing nonubiquitylated Fbw7 to copurifiy with ubiquitylated species. (C) Parkin forms lysine 48 (K48)-linked chains on Fbw7. MBP-Fbw7 from reactions analogous to those described in panel D was purified using amylose-Sepharose beads and analyzed by SDS-PAGE and Western blotting. Parallel samples were analyzed using antiubiquitin antibody or anti-K48-linked ubiquitin antibody. α, anti; Ub, ubiquitin; IP, immunoprecipitation; IB, immunoblotting. The superscript “n” indicates a variable number of ubiquitin molecules conjugated to Fbw7.

Techniques Used: Transduction, Expressing, Immunoprecipitation, SDS Page, Western Blot, In Vitro, Purification, Incubation

36) Product Images from "Clathrin light chain A drives selective myosin VI recruitment to clathrin-coated pits under membrane tension"

Article Title: Clathrin light chain A drives selective myosin VI recruitment to clathrin-coated pits under membrane tension

Journal: Nature Communications

doi: 10.1038/s41467-019-12855-6

CLCa is a direct and specific interactor of myosin VI long in triskelia and clathrin cages. a Scheme of the myosin VI highlighting the region involved in clathrin binding (amino acids 998–1131 of the long isoform). Long and short isoforms are reported together with the domains and motifs previously identified, including IQ motif, 3HB (three-helix bundle), SAH (single α-helix), MIU (motif interacting with ubiquitin), AS (alternative splicing region), and MyUb (myosin VI ubiquitin-binding domain). In orange is represented the alternatively spliced region codifying for the α2-linker 18 . b Pull-down assay with GST-CLCa and CLCb full-length and cleaved and purified fragments spanning amino acids 998–1131 of long and short myosin VI isoforms. Glutathione sepharose beads coupled to GST and GST-tagged proteins were incubated with myosin VI 998–1131 . After washes, bound proteins were eluted in Laemmli-buffer, resolved through SDS-PAGE, and stained with Coomassie. c Pull-down assay using the long and short GST-myosin VI 998–1131 constructs and brain lysates (500 μg) obtained from the indicated mouse strains. After washes, bound proteins were eluted in Laemmli-buffer, resolved through SDS-PAGE, and transferred to a nitrocellulose membrane. Immunoblot (IB) was performed with anti-clathrin heavy-chain antibody. Ponceau detect equal loading of GST proteins. d IB of the brain lysates used in ( c ), as indicated. e Co-sedimentation assay. Equimolar (1.5 μM) amount of myosin VI 998–1131 and clathrin cages were incubated at 4 °C for 45 min in the presence of detergent (0.1% Triton X-100) and then pelleted by ultracentrifugation. Precipitated proteins were dissolved in Laemmli-buffer, resolved through SDS-PAGE, and stained with Coomassie. CLCs* indicates the various CLC proteins. Note that in the native cages CLCs (CHC-CLCab) run at different molecular weight (mw) as they are from pig brain while the human CLCs used for reconstitution are bacterially produced and cleaved from GST
Figure Legend Snippet: CLCa is a direct and specific interactor of myosin VI long in triskelia and clathrin cages. a Scheme of the myosin VI highlighting the region involved in clathrin binding (amino acids 998–1131 of the long isoform). Long and short isoforms are reported together with the domains and motifs previously identified, including IQ motif, 3HB (three-helix bundle), SAH (single α-helix), MIU (motif interacting with ubiquitin), AS (alternative splicing region), and MyUb (myosin VI ubiquitin-binding domain). In orange is represented the alternatively spliced region codifying for the α2-linker 18 . b Pull-down assay with GST-CLCa and CLCb full-length and cleaved and purified fragments spanning amino acids 998–1131 of long and short myosin VI isoforms. Glutathione sepharose beads coupled to GST and GST-tagged proteins were incubated with myosin VI 998–1131 . After washes, bound proteins were eluted in Laemmli-buffer, resolved through SDS-PAGE, and stained with Coomassie. c Pull-down assay using the long and short GST-myosin VI 998–1131 constructs and brain lysates (500 μg) obtained from the indicated mouse strains. After washes, bound proteins were eluted in Laemmli-buffer, resolved through SDS-PAGE, and transferred to a nitrocellulose membrane. Immunoblot (IB) was performed with anti-clathrin heavy-chain antibody. Ponceau detect equal loading of GST proteins. d IB of the brain lysates used in ( c ), as indicated. e Co-sedimentation assay. Equimolar (1.5 μM) amount of myosin VI 998–1131 and clathrin cages were incubated at 4 °C for 45 min in the presence of detergent (0.1% Triton X-100) and then pelleted by ultracentrifugation. Precipitated proteins were dissolved in Laemmli-buffer, resolved through SDS-PAGE, and stained with Coomassie. CLCs* indicates the various CLC proteins. Note that in the native cages CLCs (CHC-CLCab) run at different molecular weight (mw) as they are from pig brain while the human CLCs used for reconstitution are bacterially produced and cleaved from GST

Techniques Used: Binding Assay, Pull Down Assay, Purification, Incubation, SDS Page, Staining, Construct, Sedimentation, Molecular Weight, Produced

CLCa:myosin VI long interact with sub-micromolar affinity. a Domain structures of CLCa and CLCb. CON conserved Hip-binding region, Hsc70 unique region in CLCa that stimulates Hsc70 activity in vitro, Ca 2+ EF-hand domain that binds calcium, CHC binding clathrin heavy chain binding region, CBD calmodulin-binding domain. Sequence conservation between the two proteins is reported below. Each line represents one amino acid, black line indicates identity. Lower panel, scheme of the selected constructs used in ( b ) together with the sequence of the three overlapping 5-carboxyfluorescein (5-FAM)-conjugated CLCa peptides used for FP analysis in ( c ). b Pull-down assay with GST-CLCa and CLCb full length and the indicated fragments of CLCa immobilized on glutathione sepharose beads and incubated with the purified fragment spanning amino acids 998–1131 of myosin VI long . After washes, bound proteins were eluted in Laemmli-buffer, resolved through SDS-PAGE, and stained with Coomassie. c FP assay using the three peptides shown in ( a ) and the purified fragment spanning amino acids 998–1131 of myosin VI long . Dissociation constants with their respective 95% confidence interval (CI) are reported in the table at the bottom. Graph is representative of three independent experiments used to calculate K d and CI. d FP assay using peptide 46–61 of CLCa and the indicated fragments of long and short myosin VI isoforms. Graph, K d , and CI as for ( c )
Figure Legend Snippet: CLCa:myosin VI long interact with sub-micromolar affinity. a Domain structures of CLCa and CLCb. CON conserved Hip-binding region, Hsc70 unique region in CLCa that stimulates Hsc70 activity in vitro, Ca 2+ EF-hand domain that binds calcium, CHC binding clathrin heavy chain binding region, CBD calmodulin-binding domain. Sequence conservation between the two proteins is reported below. Each line represents one amino acid, black line indicates identity. Lower panel, scheme of the selected constructs used in ( b ) together with the sequence of the three overlapping 5-carboxyfluorescein (5-FAM)-conjugated CLCa peptides used for FP analysis in ( c ). b Pull-down assay with GST-CLCa and CLCb full length and the indicated fragments of CLCa immobilized on glutathione sepharose beads and incubated with the purified fragment spanning amino acids 998–1131 of myosin VI long . After washes, bound proteins were eluted in Laemmli-buffer, resolved through SDS-PAGE, and stained with Coomassie. c FP assay using the three peptides shown in ( a ) and the purified fragment spanning amino acids 998–1131 of myosin VI long . Dissociation constants with their respective 95% confidence interval (CI) are reported in the table at the bottom. Graph is representative of three independent experiments used to calculate K d and CI. d FP assay using peptide 46–61 of CLCa and the indicated fragments of long and short myosin VI isoforms. Graph, K d , and CI as for ( c )

Techniques Used: Binding Assay, Activity Assay, In Vitro, Sequencing, Construct, Pull Down Assay, Incubation, Purification, SDS Page, Staining, FP Assay

37) Product Images from "Clathrin light chain A drives selective myosin VI recruitment to clathrin-coated pits under membrane tension"

Article Title: Clathrin light chain A drives selective myosin VI recruitment to clathrin-coated pits under membrane tension

Journal: Nature Communications

doi: 10.1038/s41467-019-12855-6

CLCa is a direct and specific interactor of myosin VI long in triskelia and clathrin cages. a Scheme of the myosin VI highlighting the region involved in clathrin binding (amino acids 998–1131 of the long isoform). Long and short isoforms are reported together with the domains and motifs previously identified, including IQ motif, 3HB (three-helix bundle), SAH (single α-helix), MIU (motif interacting with ubiquitin), AS (alternative splicing region), and MyUb (myosin VI ubiquitin-binding domain). In orange is represented the alternatively spliced region codifying for the α2-linker 18 . b Pull-down assay with GST-CLCa and CLCb full-length and cleaved and purified fragments spanning amino acids 998–1131 of long and short myosin VI isoforms. Glutathione sepharose beads coupled to GST and GST-tagged proteins were incubated with myosin VI 998–1131 . After washes, bound proteins were eluted in Laemmli-buffer, resolved through SDS-PAGE, and stained with Coomassie. c Pull-down assay using the long and short GST-myosin VI 998–1131 constructs and brain lysates (500 μg) obtained from the indicated mouse strains. After washes, bound proteins were eluted in Laemmli-buffer, resolved through SDS-PAGE, and transferred to a nitrocellulose membrane. Immunoblot (IB) was performed with anti-clathrin heavy-chain antibody. Ponceau detect equal loading of GST proteins. d IB of the brain lysates used in ( c ), as indicated. e Co-sedimentation assay. Equimolar (1.5 μM) amount of myosin VI 998–1131 and clathrin cages were incubated at 4 °C for 45 min in the presence of detergent (0.1% Triton X-100) and then pelleted by ultracentrifugation. Precipitated proteins were dissolved in Laemmli-buffer, resolved through SDS-PAGE, and stained with Coomassie. CLCs* indicates the various CLC proteins. Note that in the native cages CLCs (CHC-CLCab) run at different molecular weight (mw) as they are from pig brain while the human CLCs used for reconstitution are bacterially produced and cleaved from GST
Figure Legend Snippet: CLCa is a direct and specific interactor of myosin VI long in triskelia and clathrin cages. a Scheme of the myosin VI highlighting the region involved in clathrin binding (amino acids 998–1131 of the long isoform). Long and short isoforms are reported together with the domains and motifs previously identified, including IQ motif, 3HB (three-helix bundle), SAH (single α-helix), MIU (motif interacting with ubiquitin), AS (alternative splicing region), and MyUb (myosin VI ubiquitin-binding domain). In orange is represented the alternatively spliced region codifying for the α2-linker 18 . b Pull-down assay with GST-CLCa and CLCb full-length and cleaved and purified fragments spanning amino acids 998–1131 of long and short myosin VI isoforms. Glutathione sepharose beads coupled to GST and GST-tagged proteins were incubated with myosin VI 998–1131 . After washes, bound proteins were eluted in Laemmli-buffer, resolved through SDS-PAGE, and stained with Coomassie. c Pull-down assay using the long and short GST-myosin VI 998–1131 constructs and brain lysates (500 μg) obtained from the indicated mouse strains. After washes, bound proteins were eluted in Laemmli-buffer, resolved through SDS-PAGE, and transferred to a nitrocellulose membrane. Immunoblot (IB) was performed with anti-clathrin heavy-chain antibody. Ponceau detect equal loading of GST proteins. d IB of the brain lysates used in ( c ), as indicated. e Co-sedimentation assay. Equimolar (1.5 μM) amount of myosin VI 998–1131 and clathrin cages were incubated at 4 °C for 45 min in the presence of detergent (0.1% Triton X-100) and then pelleted by ultracentrifugation. Precipitated proteins were dissolved in Laemmli-buffer, resolved through SDS-PAGE, and stained with Coomassie. CLCs* indicates the various CLC proteins. Note that in the native cages CLCs (CHC-CLCab) run at different molecular weight (mw) as they are from pig brain while the human CLCs used for reconstitution are bacterially produced and cleaved from GST

Techniques Used: Binding Assay, Pull Down Assay, Purification, Incubation, SDS Page, Staining, Construct, Sedimentation, Molecular Weight, Produced

CLCa:myosin VI long interact with sub-micromolar affinity. a Domain structures of CLCa and CLCb. CON conserved Hip-binding region, Hsc70 unique region in CLCa that stimulates Hsc70 activity in vitro, Ca 2+ EF-hand domain that binds calcium, CHC binding clathrin heavy chain binding region, CBD calmodulin-binding domain. Sequence conservation between the two proteins is reported below. Each line represents one amino acid, black line indicates identity. Lower panel, scheme of the selected constructs used in ( b ) together with the sequence of the three overlapping 5-carboxyfluorescein (5-FAM)-conjugated CLCa peptides used for FP analysis in ( c ). b Pull-down assay with GST-CLCa and CLCb full length and the indicated fragments of CLCa immobilized on glutathione sepharose beads and incubated with the purified fragment spanning amino acids 998–1131 of myosin VI long . After washes, bound proteins were eluted in Laemmli-buffer, resolved through SDS-PAGE, and stained with Coomassie. c FP assay using the three peptides shown in ( a ) and the purified fragment spanning amino acids 998–1131 of myosin VI long . Dissociation constants with their respective 95% confidence interval (CI) are reported in the table at the bottom. Graph is representative of three independent experiments used to calculate K d and CI. d FP assay using peptide 46–61 of CLCa and the indicated fragments of long and short myosin VI isoforms. Graph, K d , and CI as for ( c )
Figure Legend Snippet: CLCa:myosin VI long interact with sub-micromolar affinity. a Domain structures of CLCa and CLCb. CON conserved Hip-binding region, Hsc70 unique region in CLCa that stimulates Hsc70 activity in vitro, Ca 2+ EF-hand domain that binds calcium, CHC binding clathrin heavy chain binding region, CBD calmodulin-binding domain. Sequence conservation between the two proteins is reported below. Each line represents one amino acid, black line indicates identity. Lower panel, scheme of the selected constructs used in ( b ) together with the sequence of the three overlapping 5-carboxyfluorescein (5-FAM)-conjugated CLCa peptides used for FP analysis in ( c ). b Pull-down assay with GST-CLCa and CLCb full length and the indicated fragments of CLCa immobilized on glutathione sepharose beads and incubated with the purified fragment spanning amino acids 998–1131 of myosin VI long . After washes, bound proteins were eluted in Laemmli-buffer, resolved through SDS-PAGE, and stained with Coomassie. c FP assay using the three peptides shown in ( a ) and the purified fragment spanning amino acids 998–1131 of myosin VI long . Dissociation constants with their respective 95% confidence interval (CI) are reported in the table at the bottom. Graph is representative of three independent experiments used to calculate K d and CI. d FP assay using peptide 46–61 of CLCa and the indicated fragments of long and short myosin VI isoforms. Graph, K d , and CI as for ( c )

Techniques Used: Binding Assay, Activity Assay, In Vitro, Sequencing, Construct, Pull Down Assay, Incubation, Purification, SDS Page, Staining, FP Assay

38) Product Images from "Ubiquitin charging of human class III ubiquitin-conjugating enzymes triggers their nuclear import"

Article Title: Ubiquitin charging of human class III ubiquitin-conjugating enzymes triggers their nuclear import

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200406001

The enzymatic activity of E1 is required for the interaction of UbcM2 with importin-11. (A) 35 S-importin-11–expressing reticulocyte lysates were pretreated with buffer (+ energy) or an ATP depletion mixture (hexo/glucose) for 30 min before being mixed with glutathione Sepharose beads and either GST-UbcM2 (lanes 1 and 2), GST (lanes 3 and 4), or GST-Ran (Q69L) (lanes 5 and 6). GST-Ran is marked with an asterisk. Bound and unbound proteins were resolved by SDS-PAGE and detected by CBB staining (GST fusions) or fluorography ( 35 S-importin-11). (B) 35 S-importin-11–expressing reticulocyte lysates were immunodepleted (lanes 1–4) or mock depleted (lanes 5–8) of E1 before being mixed with myc-UbcM2-H 6 (UbcM2; lanes 1, 4, 5, and 8), Ran (Q69L)-H 6 (Ran; lanes 2 and 6), or no protein (lanes 3 and 7) and Ni 2+ -agarose beads. Purified E1 was added back (lanes 4 and 8) to restore the binding interaction. Bead-associated proteins and unbound 35 S-importin-11 were analyzed by SDS-PAGE and detected by CBB staining (UbcM2, Ran) or fluorography ( 35 S-importin-11). (C) Anti-E1 Western blot of the E1 remaining (3% of lysate) in 35 S-importin-11–expressing reticulocyte lysates after immunodepletion (E1 depletion) or mock depletion (Mock depletion). Bands corresponding to E1 is indicated with an arrow, and molecular size markers are denoted to the right of the blot. (D) 35 S-importin-11–expressing reticulocyte lysates were immunodepleted of E1 and then supplemented with either buffer (lanes 1 and 4), enzymatically inactivated E1 (Iodoacet.; lanes 2 and 5), or mock-inactivated E1 (Mock; lanes 3 and 6). GST-UbcM2 is immobilized on glutathione Sepharose beads (lanes 1–3) and Ran (Q69L)-H 6 is on Ni 2+ -agarose beads (lanes 4–6). Bound proteins were detected as in B and a Western blot of the E1 added to each lysate is also shown. For A, B, and D, bound represents 50% of bead-associated proteins and unbound represents 10% of proteins remaining in lysate.
Figure Legend Snippet: The enzymatic activity of E1 is required for the interaction of UbcM2 with importin-11. (A) 35 S-importin-11–expressing reticulocyte lysates were pretreated with buffer (+ energy) or an ATP depletion mixture (hexo/glucose) for 30 min before being mixed with glutathione Sepharose beads and either GST-UbcM2 (lanes 1 and 2), GST (lanes 3 and 4), or GST-Ran (Q69L) (lanes 5 and 6). GST-Ran is marked with an asterisk. Bound and unbound proteins were resolved by SDS-PAGE and detected by CBB staining (GST fusions) or fluorography ( 35 S-importin-11). (B) 35 S-importin-11–expressing reticulocyte lysates were immunodepleted (lanes 1–4) or mock depleted (lanes 5–8) of E1 before being mixed with myc-UbcM2-H 6 (UbcM2; lanes 1, 4, 5, and 8), Ran (Q69L)-H 6 (Ran; lanes 2 and 6), or no protein (lanes 3 and 7) and Ni 2+ -agarose beads. Purified E1 was added back (lanes 4 and 8) to restore the binding interaction. Bead-associated proteins and unbound 35 S-importin-11 were analyzed by SDS-PAGE and detected by CBB staining (UbcM2, Ran) or fluorography ( 35 S-importin-11). (C) Anti-E1 Western blot of the E1 remaining (3% of lysate) in 35 S-importin-11–expressing reticulocyte lysates after immunodepletion (E1 depletion) or mock depletion (Mock depletion). Bands corresponding to E1 is indicated with an arrow, and molecular size markers are denoted to the right of the blot. (D) 35 S-importin-11–expressing reticulocyte lysates were immunodepleted of E1 and then supplemented with either buffer (lanes 1 and 4), enzymatically inactivated E1 (Iodoacet.; lanes 2 and 5), or mock-inactivated E1 (Mock; lanes 3 and 6). GST-UbcM2 is immobilized on glutathione Sepharose beads (lanes 1–3) and Ran (Q69L)-H 6 is on Ni 2+ -agarose beads (lanes 4–6). Bound proteins were detected as in B and a Western blot of the E1 added to each lysate is also shown. For A, B, and D, bound represents 50% of bead-associated proteins and unbound represents 10% of proteins remaining in lysate.

Techniques Used: Activity Assay, Expressing, SDS Page, Staining, Purification, Binding Assay, Western Blot

Importin-11 interacts in vivo with the Ub-charged forms of class III E2 enzymes. (A) Transfected HEK293T cells expressing HA 3 -importin-11 and myc-tagged UbcM2 (wt, C145A, or C145S) were harvested under nonreducing conditions and exposed to 12CA5 antibody and protein A–Sepharose beads to precipitate HA 3 -importin-11 and any associated myc-tagged UbcM2. Bead-associated and unbound proteins were separated by both nonreducing and reducing SDS-PAGE and detected by Western blotting with 12CA5-HRP (anti-HA blot) or anti-myc-HRP (anti-Myc blot) conjugates and ECL. Ub-charged UbcM2 migrates more slowly than its uncharged counterpart in nonreducing SDS-PAGE (lane 6). Under reducing conditions, Ub is readily removed from wt UbcM2, but not from the C145S mutant (wt, lanes 6 and 12 vs. C145S, lanes 5 and 11). The migration of molecular size markers is indicated to the right of the blots. (B) Lysates from transfected HEK cells expressing HA 3 -importin-11 were mixed with recombinant (C145S) UbcM2 not loaded (lanes 1 and 4) or preloaded with Ub (lanes 2, 3, 5, and 6). Precipitation of the HA 3 -importin-11 and any bound, recombinant (C145S) UbcM2 was then done as described in A, except that one lysate (lane 5) was spiked with (Q69L) Ran before 12CA5 addition. Samples were resolved by reducing SDS-PAGE, and HA 3 -importin-11 and (C145S) UbcM2 were detected with a 12CA5 antibody (Anti-HA blot) or an anti-UbcM2 antibody (Anti-UbcM2 blot), respectively. 75% of bound (lanes 4–6) and 5% of unbound (lanes 1–3) are shown. The migration of molecular size markers are indicated to the right. Ub-charged (C145S) UbcM2 (H-S-UbcM2 (C145S)~Ub) migrates more slowly than the uncharged enzyme (H-S-UbcM2 (C145S)). (C) Same experiment as in A, except that in place of the UbcM2 mutants, myc-tagged forms of UbcH6, UBE2E2, and UbcH7 were each coexpressed with HA 3 -importin-11. The Ub-charged form of each E2 is marked with an asterisk. For experiments A and C, bound represents 50% of total and unbound represents 10% of total.
Figure Legend Snippet: Importin-11 interacts in vivo with the Ub-charged forms of class III E2 enzymes. (A) Transfected HEK293T cells expressing HA 3 -importin-11 and myc-tagged UbcM2 (wt, C145A, or C145S) were harvested under nonreducing conditions and exposed to 12CA5 antibody and protein A–Sepharose beads to precipitate HA 3 -importin-11 and any associated myc-tagged UbcM2. Bead-associated and unbound proteins were separated by both nonreducing and reducing SDS-PAGE and detected by Western blotting with 12CA5-HRP (anti-HA blot) or anti-myc-HRP (anti-Myc blot) conjugates and ECL. Ub-charged UbcM2 migrates more slowly than its uncharged counterpart in nonreducing SDS-PAGE (lane 6). Under reducing conditions, Ub is readily removed from wt UbcM2, but not from the C145S mutant (wt, lanes 6 and 12 vs. C145S, lanes 5 and 11). The migration of molecular size markers is indicated to the right of the blots. (B) Lysates from transfected HEK cells expressing HA 3 -importin-11 were mixed with recombinant (C145S) UbcM2 not loaded (lanes 1 and 4) or preloaded with Ub (lanes 2, 3, 5, and 6). Precipitation of the HA 3 -importin-11 and any bound, recombinant (C145S) UbcM2 was then done as described in A, except that one lysate (lane 5) was spiked with (Q69L) Ran before 12CA5 addition. Samples were resolved by reducing SDS-PAGE, and HA 3 -importin-11 and (C145S) UbcM2 were detected with a 12CA5 antibody (Anti-HA blot) or an anti-UbcM2 antibody (Anti-UbcM2 blot), respectively. 75% of bound (lanes 4–6) and 5% of unbound (lanes 1–3) are shown. The migration of molecular size markers are indicated to the right. Ub-charged (C145S) UbcM2 (H-S-UbcM2 (C145S)~Ub) migrates more slowly than the uncharged enzyme (H-S-UbcM2 (C145S)). (C) Same experiment as in A, except that in place of the UbcM2 mutants, myc-tagged forms of UbcH6, UBE2E2, and UbcH7 were each coexpressed with HA 3 -importin-11. The Ub-charged form of each E2 is marked with an asterisk. For experiments A and C, bound represents 50% of total and unbound represents 10% of total.

Techniques Used: In Vivo, Transfection, Expressing, SDS Page, Western Blot, Mutagenesis, Migration, Recombinant

The active site cysteine of UbcM2 is required for the interaction with importin-11. (A) HF7c (MAT a ) yeast expressing the indicated bait proteins (on left) as GAL4-DBD fusions were mated with W303 α (MAT α ) strains expressing VP16 TA domain fusions (across top). Diploid yeast were selected on Leu − /Trp − plates and replica-plated onto Leu − /Trp − /His − plates with (L,W,H − + 3-AT) or without (L,W,H − ) 3-amino triazole (3-AT). (B) Wt (lane 1), C145S (lane 2), or C145A (lane 3) UbcM2 fused to two GFPs and 6× His tag (UbcM2-GGH 6 ) were mixed with Ni 2+ -agarose beads and a reticuloctye lysate containing 35 S-labeled importin-11. Bound proteins (50% of bound) were separated by SDS-PAGE and detected by CBB staining (UbcM2-GGH 6 ) or fluorography ( 35 S-importin-11).
Figure Legend Snippet: The active site cysteine of UbcM2 is required for the interaction with importin-11. (A) HF7c (MAT a ) yeast expressing the indicated bait proteins (on left) as GAL4-DBD fusions were mated with W303 α (MAT α ) strains expressing VP16 TA domain fusions (across top). Diploid yeast were selected on Leu − /Trp − plates and replica-plated onto Leu − /Trp − /His − plates with (L,W,H − + 3-AT) or without (L,W,H − ) 3-amino triazole (3-AT). (B) Wt (lane 1), C145S (lane 2), or C145A (lane 3) UbcM2 fused to two GFPs and 6× His tag (UbcM2-GGH 6 ) were mixed with Ni 2+ -agarose beads and a reticuloctye lysate containing 35 S-labeled importin-11. Bound proteins (50% of bound) were separated by SDS-PAGE and detected by CBB staining (UbcM2-GGH 6 ) or fluorography ( 35 S-importin-11).

Techniques Used: Expressing, Labeling, SDS Page, Staining

39) Product Images from "A secreted Ustilago maydis effector promotes virulence by targeting anthocyanin biosynthesis in maize"

Article Title: A secreted Ustilago maydis effector promotes virulence by targeting anthocyanin biosynthesis in maize

Journal: eLife

doi: 10.7554/eLife.01355

Physical interaction of Tin2 protein with full-length ZmTTK1 and in-vitro kinase activity of ZmTTK1. ( A ) Physical interaction of Tin2 and ZmTTK1 demonstrated by in vitro GST-pull down assay using recombinant proteins. Recombinant GST-Tin2 26-207 or GST-Tin2 26-202 protein bound to glutathione sepharose beads (GE healthcare), respectively, was incubated with extract from induced BL21 (DE3)/pPRIBA102-ZmTTK1 at 4°C for 1 hr. GST fusion proteins were eluted with reduced glutathione. Strep-ZmTTK1 in eluate was detected by western blot (WB) using Strep-tactin-HRP and α-GST antibody for detection, respectively. Top panel detects precipitated Strep-ZmTTK1, bottom panel detects input GST-Tin2 fusion proteins. ( B ) Full-length recombinant Strep-ZmTTK1 expressed and purified from E. coli was detected by western blot analysis with Strep-tactin-HRP. ( C ) In vitro kinase activity of recombinant Strep-ZmTTK1 protein. Recombinant GST-Tin2 26-207 and Strep-ZmTTK1 proteins at indicated concentration were pre-incubated for 60 min at 4°C followed by addition of γ- 32 P ATP and myelin basic protein (MBP). Bovine serum albumin (BSA) was used as a control. Incubation continued at 28°C for 30 min. Proteins were separated by SDS-PAGE and phosphorylated protein was detected. DOI: http://dx.doi.org/10.7554/eLife.01355.016
Figure Legend Snippet: Physical interaction of Tin2 protein with full-length ZmTTK1 and in-vitro kinase activity of ZmTTK1. ( A ) Physical interaction of Tin2 and ZmTTK1 demonstrated by in vitro GST-pull down assay using recombinant proteins. Recombinant GST-Tin2 26-207 or GST-Tin2 26-202 protein bound to glutathione sepharose beads (GE healthcare), respectively, was incubated with extract from induced BL21 (DE3)/pPRIBA102-ZmTTK1 at 4°C for 1 hr. GST fusion proteins were eluted with reduced glutathione. Strep-ZmTTK1 in eluate was detected by western blot (WB) using Strep-tactin-HRP and α-GST antibody for detection, respectively. Top panel detects precipitated Strep-ZmTTK1, bottom panel detects input GST-Tin2 fusion proteins. ( B ) Full-length recombinant Strep-ZmTTK1 expressed and purified from E. coli was detected by western blot analysis with Strep-tactin-HRP. ( C ) In vitro kinase activity of recombinant Strep-ZmTTK1 protein. Recombinant GST-Tin2 26-207 and Strep-ZmTTK1 proteins at indicated concentration were pre-incubated for 60 min at 4°C followed by addition of γ- 32 P ATP and myelin basic protein (MBP). Bovine serum albumin (BSA) was used as a control. Incubation continued at 28°C for 30 min. Proteins were separated by SDS-PAGE and phosphorylated protein was detected. DOI: http://dx.doi.org/10.7554/eLife.01355.016

Techniques Used: In Vitro, Activity Assay, Pull Down Assay, Recombinant, Incubation, Western Blot, Purification, Concentration Assay, SDS Page

Tin2 protein stabilizes cytoplasmic maize protein kinase ZmTTK1. ( A ) ZmTTK1-HA and HA-ZmTTK1 expression in N. benthamiana . HA-ZmTTK1 or ZmTTK1-HA protein was transiently expressed in N. benthamiana after infiltration with the respective A. tumefaciens strains GV3101 carrying pBIN19AN-HA-ZmTTK1 and pBIN19AN-ZmTTK1-HA. These plasmids express the indicated fusion proteins with a C-terminal IgG binding site. Expression was shown by western blot (WB) using indicated antibody. Asterisk labels a non-specific band. Rubisco large subunit (RBCL) stained with coomassie brilliant blue (CBB) served as a loading control. ZmTTK1-HA transcripts were analyzed by RT-PCR, EF1α served as a control. ( B ) Protein expression of ZmTTK1-HA with proteasome inhibitor MG132 (100 µM) and ZmTTK1 S279/282A -HA. ( C ) Detection of poly-ubiquitinated ZmTTK1. After immunoprecipitation with human IgG-agarose, proteins were subjected to western blot to detect poly-ubiquitinated ZmTTK1 protein (arrow). The western blot was developed with monoclonal anti-ubiquitin antibody (Sigma-Aldrich). ( D ) Co-expression of ZmTTK1-HA with myc-Tin2. Total protein was analyzed by western blot using indicated antibodies. ( E ) Immunoprecipitated ZmTTK1-HA protein from ( D ) was analyzed by western blot. Kinase activity of immunoprecipitated samples shown on top was analyzed using MBP as a substrate (bottom). DOI: http://dx.doi.org/10.7554/eLife.01355.017
Figure Legend Snippet: Tin2 protein stabilizes cytoplasmic maize protein kinase ZmTTK1. ( A ) ZmTTK1-HA and HA-ZmTTK1 expression in N. benthamiana . HA-ZmTTK1 or ZmTTK1-HA protein was transiently expressed in N. benthamiana after infiltration with the respective A. tumefaciens strains GV3101 carrying pBIN19AN-HA-ZmTTK1 and pBIN19AN-ZmTTK1-HA. These plasmids express the indicated fusion proteins with a C-terminal IgG binding site. Expression was shown by western blot (WB) using indicated antibody. Asterisk labels a non-specific band. Rubisco large subunit (RBCL) stained with coomassie brilliant blue (CBB) served as a loading control. ZmTTK1-HA transcripts were analyzed by RT-PCR, EF1α served as a control. ( B ) Protein expression of ZmTTK1-HA with proteasome inhibitor MG132 (100 µM) and ZmTTK1 S279/282A -HA. ( C ) Detection of poly-ubiquitinated ZmTTK1. After immunoprecipitation with human IgG-agarose, proteins were subjected to western blot to detect poly-ubiquitinated ZmTTK1 protein (arrow). The western blot was developed with monoclonal anti-ubiquitin antibody (Sigma-Aldrich). ( D ) Co-expression of ZmTTK1-HA with myc-Tin2. Total protein was analyzed by western blot using indicated antibodies. ( E ) Immunoprecipitated ZmTTK1-HA protein from ( D ) was analyzed by western blot. Kinase activity of immunoprecipitated samples shown on top was analyzed using MBP as a substrate (bottom). DOI: http://dx.doi.org/10.7554/eLife.01355.017

Techniques Used: Expressing, Binding Assay, Western Blot, Staining, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation, Activity Assay

40) Product Images from "Protein tyrosine phosphatase SAP-1 protects against colitis through regulation of CEACAM20 in the intestinal epithelium"

Article Title: Protein tyrosine phosphatase SAP-1 protects against colitis through regulation of CEACAM20 in the intestinal epithelium

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1510167112

Tyrosine-phosphorylated CEACAM20 binds to the SH2 domains of Syk and no interaction of CEACAM20 with c-Src, Fyn, or Frk. ( A ) Lysates of pervanadate-treated HEK293A cells expressing MycCC20(WT) or MycCC20(2YF) were incubated with GST or a GST fusion protein containing the SH2 domains of Syk immobilized to glutathione-agarose beads, after which the bead-bound proteins were subjected to immunoblot analysis with antibodies to CEACAM20 ( Upper ). The same amount of the lysates (input) was also subjected directly to immunoblot analysis ( Lower Left ). The purity of the GST proteins used in the assay was demonstrated by SDS/PAGE and staining with Coomassie brilliant blue (CBB) ( Lower Right ). ( B – D ) Lysates of HEK293A cells transfected with expression vectors for the indicated proteins were subjected to immunoprecipitation with antibodies to Myc, and the resulting precipitates as well as the original cell lysates were subjected to immunoblot analysis with the indicated antibodies. All data are representative of three independent experiments.
Figure Legend Snippet: Tyrosine-phosphorylated CEACAM20 binds to the SH2 domains of Syk and no interaction of CEACAM20 with c-Src, Fyn, or Frk. ( A ) Lysates of pervanadate-treated HEK293A cells expressing MycCC20(WT) or MycCC20(2YF) were incubated with GST or a GST fusion protein containing the SH2 domains of Syk immobilized to glutathione-agarose beads, after which the bead-bound proteins were subjected to immunoblot analysis with antibodies to CEACAM20 ( Upper ). The same amount of the lysates (input) was also subjected directly to immunoblot analysis ( Lower Left ). The purity of the GST proteins used in the assay was demonstrated by SDS/PAGE and staining with Coomassie brilliant blue (CBB) ( Lower Right ). ( B – D ) Lysates of HEK293A cells transfected with expression vectors for the indicated proteins were subjected to immunoprecipitation with antibodies to Myc, and the resulting precipitates as well as the original cell lysates were subjected to immunoblot analysis with the indicated antibodies. All data are representative of three independent experiments.

Techniques Used: Expressing, Incubation, SDS Page, Staining, Transfection, Immunoprecipitation

Identification of CEACAM20 as a tyrosine-phosphorylated protein in the intestinal epithelium of SAP-1–deficient mice. ( A ) Sections of the colon of 10-wk-old WT or Sap1 −/− mice were stained with antibodies to phosphotyrosine (pY, red) and with DAPI (blue). (Scale bar, 20 μm.) Arrowheads indicate prominent staining for phosphotyrosine along the apical surface of the colonic epithelium in the mutant. ( B ) Microvillus membranes prepared from the entire small intestine of WT or Sap1 −/− mice were subjected to immunoblot analysis with antibodies to phosphotyrosine (α-pY), to SAP-1, or to β-actin ( Left ). Bands corresponding to proteins whose level of tyrosine phosphorylation was markedly increased in Sap1 −/− mice are indicated by arrowheads. Tyrosine-phosphorylated proteins purified from a solubilized microvillus membrane fraction of Sap1 −/− mice with the use of agarose-bead–conjugated antibodies to phosphotyrosine were fractionated by SDS/PAGE and visualized by silver staining ( Right ). The protein bands indicated by the asterisks were analyzed by MS. The ∼100-, ∼60-, and ∼40-kDa protein bands (***, **, and *) contained the indicated proteins. ( C ) Schematic representation of the structure of mouse CEACAM20 showing four Ig-like domains in the extracellular region and four potential tyrosine phosphorylation sites, two of which constitute an ITAM, in the cytoplasmic region. ( D ) Microvillus membranes prepared from the entire small intestine of WT or Sap1 −/− mice were subjected to immunoprecipitation (IP) with antibodies to CEACAM20 (α-CC20) or to Eps8, and the resulting precipitates were subjected to immunoblot analysis of phosphotyrosine, CEACAM20, or Eps8. Data are representative of three ( A ) or two ( B and D ) independent experiments.
Figure Legend Snippet: Identification of CEACAM20 as a tyrosine-phosphorylated protein in the intestinal epithelium of SAP-1–deficient mice. ( A ) Sections of the colon of 10-wk-old WT or Sap1 −/− mice were stained with antibodies to phosphotyrosine (pY, red) and with DAPI (blue). (Scale bar, 20 μm.) Arrowheads indicate prominent staining for phosphotyrosine along the apical surface of the colonic epithelium in the mutant. ( B ) Microvillus membranes prepared from the entire small intestine of WT or Sap1 −/− mice were subjected to immunoblot analysis with antibodies to phosphotyrosine (α-pY), to SAP-1, or to β-actin ( Left ). Bands corresponding to proteins whose level of tyrosine phosphorylation was markedly increased in Sap1 −/− mice are indicated by arrowheads. Tyrosine-phosphorylated proteins purified from a solubilized microvillus membrane fraction of Sap1 −/− mice with the use of agarose-bead–conjugated antibodies to phosphotyrosine were fractionated by SDS/PAGE and visualized by silver staining ( Right ). The protein bands indicated by the asterisks were analyzed by MS. The ∼100-, ∼60-, and ∼40-kDa protein bands (***, **, and *) contained the indicated proteins. ( C ) Schematic representation of the structure of mouse CEACAM20 showing four Ig-like domains in the extracellular region and four potential tyrosine phosphorylation sites, two of which constitute an ITAM, in the cytoplasmic region. ( D ) Microvillus membranes prepared from the entire small intestine of WT or Sap1 −/− mice were subjected to immunoprecipitation (IP) with antibodies to CEACAM20 (α-CC20) or to Eps8, and the resulting precipitates were subjected to immunoblot analysis of phosphotyrosine, CEACAM20, or Eps8. Data are representative of three ( A ) or two ( B and D ) independent experiments.

Techniques Used: Mouse Assay, Staining, Mutagenesis, Purification, SDS Page, Silver Staining, Mass Spectrometry, Immunoprecipitation

41) Product Images from "Regulation of Tsg101 Expression by the Steadiness Box: A Role of Tsg101-associated Ligase"

Article Title: Regulation of Tsg101 Expression by the Steadiness Box: A Role of Tsg101-associated Ligase

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E07-09-0957

Mapping the determinants for Tal-mediated polyubiquitination of Tsg101. (A) Coexpression of wild-type YFP-Tsg101, YFP-Tsg101 M95A , or YFP-Tsg101 1-360 in the presence of GST-Tal or GST empty vector in 293T cells. Cell lysates were harvested 24 h after transfection and Tsg101 expression was determined by Western blot using α-GFP antibody. Lysates were also probed with α-Hsp90 as a loading control. (B) Pulldown assay to measure relative ubiquitination of wild-type and mutant/truncated GST-Tsg101 by YFP-Tal. GST-Tsg101 constructs were transfected in the presence or absence of YFP-Tal, with HA-ubiquitin in 293T cells. Forty-eight hours after transfection, cell lysates were precipitated with glutathione-Sepharose beads, and the bound fractions were analyzed by immunoblotting with α-Tsg101 antibody. Samples were normalized for equal Tsg101 expression and reprobed with α-Tsg101 (left), α-poly-ubiquitin (middle), and α-HA (right) antibodies.
Figure Legend Snippet: Mapping the determinants for Tal-mediated polyubiquitination of Tsg101. (A) Coexpression of wild-type YFP-Tsg101, YFP-Tsg101 M95A , or YFP-Tsg101 1-360 in the presence of GST-Tal or GST empty vector in 293T cells. Cell lysates were harvested 24 h after transfection and Tsg101 expression was determined by Western blot using α-GFP antibody. Lysates were also probed with α-Hsp90 as a loading control. (B) Pulldown assay to measure relative ubiquitination of wild-type and mutant/truncated GST-Tsg101 by YFP-Tal. GST-Tsg101 constructs were transfected in the presence or absence of YFP-Tal, with HA-ubiquitin in 293T cells. Forty-eight hours after transfection, cell lysates were precipitated with glutathione-Sepharose beads, and the bound fractions were analyzed by immunoblotting with α-Tsg101 antibody. Samples were normalized for equal Tsg101 expression and reprobed with α-Tsg101 (left), α-poly-ubiquitin (middle), and α-HA (right) antibodies.

Techniques Used: Plasmid Preparation, Transfection, Expressing, Western Blot, Mutagenesis, Construct

VPS28 protects C-terminal lysine targets of Tal-mediated polyubiquitination. (A) Coexpression experiment to compare the ability of Tsg101-binding proteins VSP28 and Cep55 to prevent Tsg101 degradation by Tal. Plasmids encoding GST-Tsg101 with GST-Tal or GST-empty vector and empty control vector, YFP-VPS28 or YFP-Cep55, were cotransfected in 293T cells. Cells were lysed 24 h after transfection and analyzed by immunoblotting with α-Tsg101, α-GFP, and α-Hsp90 antibodies. (B) Coprecipitation experiment demonstrating that VPS28 blocks Tsg101 ubiquitination. GST-Tsg101 and HA-ubiquitin expressed in 293T cells in the presence or absence of YFP-Tal and Myc-VPS28 as indicated. Cell lysates were subjected to precipitation with glutathione-Sepharose beads and analyzed by Western blot, with α-Tsg101 to normalize for Tsg101 expression before reprobing with α-Tsg101 and α-HA monoclonal antibodies. (C) Coexpression of wild-type YFP-Tsg101, YFP-Tsg101 lysine to arginine mutants (progressively mutating lysines 3′ to 5′ from C-terminus), or YFP-Tsg101 M95A , with GST-Tal or GST-empty and Myc-empty or Myc-VPS28 as indicated. Cells were lysed 24 h after transfection and analyzed by Western blot using α-GFP antibody. (D) Densitometry of Western blot shown in C. Expression of Tsg101 in the presence of Tal, with or without VPS28 is shown as a percentage of the control (lane 2) for each construct (n = 3 ± SD). (E) Confocal microscopy images of HeLa cells expressing YFP-Tal and Cherry-Tsg101 with CFP-VPS28 (top panels), or CFP-empty vector (bottom panels) as indicated.
Figure Legend Snippet: VPS28 protects C-terminal lysine targets of Tal-mediated polyubiquitination. (A) Coexpression experiment to compare the ability of Tsg101-binding proteins VSP28 and Cep55 to prevent Tsg101 degradation by Tal. Plasmids encoding GST-Tsg101 with GST-Tal or GST-empty vector and empty control vector, YFP-VPS28 or YFP-Cep55, were cotransfected in 293T cells. Cells were lysed 24 h after transfection and analyzed by immunoblotting with α-Tsg101, α-GFP, and α-Hsp90 antibodies. (B) Coprecipitation experiment demonstrating that VPS28 blocks Tsg101 ubiquitination. GST-Tsg101 and HA-ubiquitin expressed in 293T cells in the presence or absence of YFP-Tal and Myc-VPS28 as indicated. Cell lysates were subjected to precipitation with glutathione-Sepharose beads and analyzed by Western blot, with α-Tsg101 to normalize for Tsg101 expression before reprobing with α-Tsg101 and α-HA monoclonal antibodies. (C) Coexpression of wild-type YFP-Tsg101, YFP-Tsg101 lysine to arginine mutants (progressively mutating lysines 3′ to 5′ from C-terminus), or YFP-Tsg101 M95A , with GST-Tal or GST-empty and Myc-empty or Myc-VPS28 as indicated. Cells were lysed 24 h after transfection and analyzed by Western blot using α-GFP antibody. (D) Densitometry of Western blot shown in C. Expression of Tsg101 in the presence of Tal, with or without VPS28 is shown as a percentage of the control (lane 2) for each construct (n = 3 ± SD). (E) Confocal microscopy images of HeLa cells expressing YFP-Tal and Cherry-Tsg101 with CFP-VPS28 (top panels), or CFP-empty vector (bottom panels) as indicated.

Techniques Used: Binding Assay, Plasmid Preparation, Transfection, Western Blot, Expressing, Construct, Confocal Microscopy

Tal specifically degrades Tsg101 via conjugation of polyubiquitin chains. (A) YFP-Tsg101 or YFP-Tsg101 157-390 fusion proteins were coexpressed in 293T cells with decreasing amounts of YFP-Tal. Samples were lysed 24 h after transfection and analyzed by immunoblotting with an α-GFP mAb. (B) Coexpression of YFP-Tsg101 fusion protein with GST, GST-fused full-length Tal, or Tal deletions as indicated. Tal 1-648 lacks the double PTAP-PSAP motif and RING domain, Tal 1-674 lacks the RING domain only and Tal δPTAP lacks the PTAP-PSAP motif. Twenty-four hours after transfection cells were lysed and analyzed by Western blot. YFP-Tsg101 expression was detected by immunoblotting with an α-GFP mAb and GST-Tal expression was determined using an α-Tal antibody raised against the N-terminus of Tal. (C) Comparison of GST-Tsg101 expression in the presence of YFP-Tal, YFP-Mahogunin, or empty vector control. Transfected cells were lysed 24 h after transfection and analyzed by Western blot. GST-Tsg101 or YFP- fusion protein expression was detected using α-Tsg101 and α-GFP antibodies, respectively. Lysates were probed with α-Hsp90 as a loading control. (D) Coprecipitation experiment to determine relative ubiquitination of Tsg101 by full-length or mutant Tal. 293T cells were cotransfected with plasmids encoding YFP-Tal, YFP-Tal δPTAP , or empty vector with GST-Tsg101 and HA-ubiquitin. Forty-eight hours after transfection cell lysates were precipitated with glutathione-Sepharose beads, and the bound fractions were analyzed by immunoblotting with α-Tsg101 antibody. Samples were normalized for equal Tsg101 expression and probed with α-Tsg101 and α-HA antibodies. YFP-Tal and YFP-Tal δPTAP expression in cell lysates was determined using α-GFP antibody. (E) Comparison of GST-Tsg101 polyubiquitination in the presence of YFP-Tal, YFP-Mahogunin or empty vector control. Cell lysates were subjected to precipitation with glutathione-Sepharose beads as before, and analyzed by Western blot. Samples were normalized for equal GST-Tsg101 expression before rerunning and probing with α-Tsg101 and α-polyubiquitin antibodies.
Figure Legend Snippet: Tal specifically degrades Tsg101 via conjugation of polyubiquitin chains. (A) YFP-Tsg101 or YFP-Tsg101 157-390 fusion proteins were coexpressed in 293T cells with decreasing amounts of YFP-Tal. Samples were lysed 24 h after transfection and analyzed by immunoblotting with an α-GFP mAb. (B) Coexpression of YFP-Tsg101 fusion protein with GST, GST-fused full-length Tal, or Tal deletions as indicated. Tal 1-648 lacks the double PTAP-PSAP motif and RING domain, Tal 1-674 lacks the RING domain only and Tal δPTAP lacks the PTAP-PSAP motif. Twenty-four hours after transfection cells were lysed and analyzed by Western blot. YFP-Tsg101 expression was detected by immunoblotting with an α-GFP mAb and GST-Tal expression was determined using an α-Tal antibody raised against the N-terminus of Tal. (C) Comparison of GST-Tsg101 expression in the presence of YFP-Tal, YFP-Mahogunin, or empty vector control. Transfected cells were lysed 24 h after transfection and analyzed by Western blot. GST-Tsg101 or YFP- fusion protein expression was detected using α-Tsg101 and α-GFP antibodies, respectively. Lysates were probed with α-Hsp90 as a loading control. (D) Coprecipitation experiment to determine relative ubiquitination of Tsg101 by full-length or mutant Tal. 293T cells were cotransfected with plasmids encoding YFP-Tal, YFP-Tal δPTAP , or empty vector with GST-Tsg101 and HA-ubiquitin. Forty-eight hours after transfection cell lysates were precipitated with glutathione-Sepharose beads, and the bound fractions were analyzed by immunoblotting with α-Tsg101 antibody. Samples were normalized for equal Tsg101 expression and probed with α-Tsg101 and α-HA antibodies. YFP-Tal and YFP-Tal δPTAP expression in cell lysates was determined using α-GFP antibody. (E) Comparison of GST-Tsg101 polyubiquitination in the presence of YFP-Tal, YFP-Mahogunin or empty vector control. Cell lysates were subjected to precipitation with glutathione-Sepharose beads as before, and analyzed by Western blot. Samples were normalized for equal GST-Tsg101 expression before rerunning and probing with α-Tsg101 and α-polyubiquitin antibodies.

Techniques Used: Conjugation Assay, Transfection, Western Blot, Expressing, Plasmid Preparation, Mutagenesis

42) Product Images from "Inhibitory Role of Plk1 in the Regulation of p73-dependent Apoptosis through Physical Interaction and Phosphorylation *Inhibitory Role of Plk1 in the Regulation of p73-dependent Apoptosis through Physical Interaction and Phosphorylation * S⃞"

Article Title: Inhibitory Role of Plk1 in the Regulation of p73-dependent Apoptosis through Physical Interaction and Phosphorylation *Inhibitory Role of Plk1 in the Regulation of p73-dependent Apoptosis through Physical Interaction and Phosphorylation * S⃞

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M710608200

Kinase domain of Plk1 is essential for the interaction with p73. A , schematic drawing of wild-type Plk1 and its deletion mutants. KD , kinase domain; PB , polo-box domain. B , Coomassie Brilliant Blue staining of GST-p73α-(1–130) used for this study. C, in vitro pulldown assay. Equal amount of GST-p73α-(1–130) was incubated with radiolabeled FLAG-Plk1 deletion mutants ( left panel ). After incubation, GST-p73α-(1–130) was precipitated by glutathione-Sepharose beads, and bound materials were separated by SDS-PAGE followed by autoradiography.
Figure Legend Snippet: Kinase domain of Plk1 is essential for the interaction with p73. A , schematic drawing of wild-type Plk1 and its deletion mutants. KD , kinase domain; PB , polo-box domain. B , Coomassie Brilliant Blue staining of GST-p73α-(1–130) used for this study. C, in vitro pulldown assay. Equal amount of GST-p73α-(1–130) was incubated with radiolabeled FLAG-Plk1 deletion mutants ( left panel ). After incubation, GST-p73α-(1–130) was precipitated by glutathione-Sepharose beads, and bound materials were separated by SDS-PAGE followed by autoradiography.

Techniques Used: Staining, In Vitro, Incubation, SDS Page, Autoradiography

NH 2 -terminal small domain of p73 is required for the interaction with Plk1. A , domain structure of wild-type p73α and schematic representation of GST-tagged p73α deletion mutants. TA , transactivation domain; DB , DNA-binding domain; OD , oligomerization domain; SAM , sterileα-motif domain. Numbers indicate amino acid positions. B , GST and GST-p73α fusion proteins were purified by glutathione-Sepharose beads and separated by 10% SDS-PAGE followed by Coomassie Brilliant Blue staining. C, in vitro pulldown assay. Equal amount of radiolabeled FLAG-Plk1 was incubated with GST or with the indicated GST-p73α fusion proteins. After incubation, GST or GST-p73α fusion proteins were recovered by glutathione-Sepharose beads, and bound materials were resolved by 10% SDS-PAGE followed by autoradiography.
Figure Legend Snippet: NH 2 -terminal small domain of p73 is required for the interaction with Plk1. A , domain structure of wild-type p73α and schematic representation of GST-tagged p73α deletion mutants. TA , transactivation domain; DB , DNA-binding domain; OD , oligomerization domain; SAM , sterileα-motif domain. Numbers indicate amino acid positions. B , GST and GST-p73α fusion proteins were purified by glutathione-Sepharose beads and separated by 10% SDS-PAGE followed by Coomassie Brilliant Blue staining. C, in vitro pulldown assay. Equal amount of radiolabeled FLAG-Plk1 was incubated with GST or with the indicated GST-p73α fusion proteins. After incubation, GST or GST-p73α fusion proteins were recovered by glutathione-Sepharose beads, and bound materials were resolved by 10% SDS-PAGE followed by autoradiography.

Techniques Used: Binding Assay, Purification, SDS Page, Staining, In Vitro, Incubation, Autoradiography

Plk1 has an ability to phosphorylate p73 at its NH 2 -terminal region in vitro . A , Coomassie Brilliant Blue staining of GST or GST-p73α fusion proteins used for in vitro kinase reaction. B, in vitro kinase assay. GST or the indicated GST-p73α deletion mutants bound to glutathione-Sepharose beads were washed with washing buffer and resuspended in 90 μl of kinase reaction buffer. Protein X, which was supplied by manufacturer, was used as a positive control. 10 μl of the active form of Plk1 were added to the reaction mixtures and incubated at 30 °C for 30 min. The reaction mixtures were washed with washing buffer and then incubated with 100 μl of polyclonal anti-phospho-Thr antibody at room temperature for 30 min followed by incubation with horseradish peroxidase-conjugated anti-rabbit IgG at room temperature for 30 min. After incubation, 100 μl of substrate reagent were added to the reaction mixtures and incubated at room temperature for 5 min. Yellow coloration indicates the Plk1-mediated phosphorylation at Thr residue. After the addition of 100 μl of stop solution, supernatant was transferred into 96-well tissue culture plate, and the absorbance reading was carried out at 450/540 nm using the microplate reader ( C ).
Figure Legend Snippet: Plk1 has an ability to phosphorylate p73 at its NH 2 -terminal region in vitro . A , Coomassie Brilliant Blue staining of GST or GST-p73α fusion proteins used for in vitro kinase reaction. B, in vitro kinase assay. GST or the indicated GST-p73α deletion mutants bound to glutathione-Sepharose beads were washed with washing buffer and resuspended in 90 μl of kinase reaction buffer. Protein X, which was supplied by manufacturer, was used as a positive control. 10 μl of the active form of Plk1 were added to the reaction mixtures and incubated at 30 °C for 30 min. The reaction mixtures were washed with washing buffer and then incubated with 100 μl of polyclonal anti-phospho-Thr antibody at room temperature for 30 min followed by incubation with horseradish peroxidase-conjugated anti-rabbit IgG at room temperature for 30 min. After incubation, 100 μl of substrate reagent were added to the reaction mixtures and incubated at room temperature for 5 min. Yellow coloration indicates the Plk1-mediated phosphorylation at Thr residue. After the addition of 100 μl of stop solution, supernatant was transferred into 96-well tissue culture plate, and the absorbance reading was carried out at 450/540 nm using the microplate reader ( C ).

Techniques Used: In Vitro, Staining, Kinase Assay, Positive Control, Incubation

43) Product Images from "The Chlamydia trachomatis Type III Secretion Chaperone Slc1 Engages Multiple Early Effectors, Including TepP, a Tyrosine-phosphorylated Protein Required for the Recruitment of CrkI-II to Nascent Inclusions and Innate Immune Signaling"

Article Title: The Chlamydia trachomatis Type III Secretion Chaperone Slc1 Engages Multiple Early Effectors, Including TepP, a Tyrosine-phosphorylated Protein Required for the Recruitment of CrkI-II to Nascent Inclusions and Innate Immune Signaling

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1003954

Slc1 associates as stable, multi-protein complexes with TARP, Ct694, Ct695 and Ct875/TepP, and enhances their secretion via the T3S system. A–B) Slc1 binds its putative target effectors in vitro . Slc1 was co-expressed in E. coli with GST-tagged TARP, Ct694, Ct695 and Ct875/TepP fusion proteins. The GST-tagged proteins were isolated from cell lysates with glutathione sepharose beads, and the relative levels of Slc1 co-isolated was assessed by immunoblot analysis (A). GST and GST-288 served as negative controls. To assess the relative size of these complexes, TARP, Ct694, Ct695 and Ct875/TepP were fused to a hexahistidine tag and co-expressed with Slc1. Bound proteins were purified using a Nickel resin, eluted with 500 mM Imidazole, and analyzed by gel filtration chromatography (B). Fraction numbers are provided on the top. Molecular size markers: Alcohol Dehydrogenase (150 kDa), Conalbumin (75 kDa) and Carbonic Anhydrase (29 kDa), peaked at F16-17, F20-21 and F26, respectively. No peak was observed between fractions 8–20 in Slc1-6xHis sample in the absence of co-expressed effectors. C ) Slc1 enhances the T3S-dependent secretion of Ct694, Ct695 and Ct875/TepP. Y. pestis KIM8-E (Δ ail ) was co-transformed with plasmids expressing Ct694, Ct875/TepP and FLAG-tagged Ct695 and untagged Slc1 or Mcsc in the combinations shown. T3S was induced by calcium chelation and the relative amount of protein secreted into the supernatants was assessed by quantitative immunoblots. Sup-cell free supernatant. Mcsc did not enhance the secretion of effectors, indicating that the secretion chaperone activity of Slc1 is specific for its target substrates.
Figure Legend Snippet: Slc1 associates as stable, multi-protein complexes with TARP, Ct694, Ct695 and Ct875/TepP, and enhances their secretion via the T3S system. A–B) Slc1 binds its putative target effectors in vitro . Slc1 was co-expressed in E. coli with GST-tagged TARP, Ct694, Ct695 and Ct875/TepP fusion proteins. The GST-tagged proteins were isolated from cell lysates with glutathione sepharose beads, and the relative levels of Slc1 co-isolated was assessed by immunoblot analysis (A). GST and GST-288 served as negative controls. To assess the relative size of these complexes, TARP, Ct694, Ct695 and Ct875/TepP were fused to a hexahistidine tag and co-expressed with Slc1. Bound proteins were purified using a Nickel resin, eluted with 500 mM Imidazole, and analyzed by gel filtration chromatography (B). Fraction numbers are provided on the top. Molecular size markers: Alcohol Dehydrogenase (150 kDa), Conalbumin (75 kDa) and Carbonic Anhydrase (29 kDa), peaked at F16-17, F20-21 and F26, respectively. No peak was observed between fractions 8–20 in Slc1-6xHis sample in the absence of co-expressed effectors. C ) Slc1 enhances the T3S-dependent secretion of Ct694, Ct695 and Ct875/TepP. Y. pestis KIM8-E (Δ ail ) was co-transformed with plasmids expressing Ct694, Ct875/TepP and FLAG-tagged Ct695 and untagged Slc1 or Mcsc in the combinations shown. T3S was induced by calcium chelation and the relative amount of protein secreted into the supernatants was assessed by quantitative immunoblots. Sup-cell free supernatant. Mcsc did not enhance the secretion of effectors, indicating that the secretion chaperone activity of Slc1 is specific for its target substrates.

Techniques Used: In Vitro, Isolation, Purification, Filtration, Chromatography, Transformation Assay, Expressing, Western Blot, Activity Assay

A distinct set of Chlamydia proteins associate with Slc1 in the Elementary Body (EB) form. A ) Relative protein mass composition of the C. trachomatis EB. Approximately 2% of total EB protein mass is comprised of T3S chaperones, with Slc1, Scc2 and Mcsc accounting for over 99% of their mass. This figure represents a reanalysis of data reported in [31] . B ) Protein interaction network for Slc1 and Mcsc. Cell lysates from gradient purified EBs were incubated with anti-Slc1 antibodies, anti-Mcsc antibodies, or non-specific IgG cross-linked to an agarose resin. Bound proteins were eluted at low pH, digested with trypsin and identified by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) ( Table S1 ). Only proteins that displayed specific interactions are shown. C ) Specificity of Slc1-interactions. EB lysates were incubated with either anti-Slc1 antisera or pre-immune sera, and bound proteins were captured on Protein A/G agarose resin. The eluate (bound) and flow through (unbound) from both Slc1 IP and control IP were analyzed by immunoblotting with antibodies against selected proteins. MOMP is an abundant Chlamydia protein that serves as control for the specificity of the interactions shown.
Figure Legend Snippet: A distinct set of Chlamydia proteins associate with Slc1 in the Elementary Body (EB) form. A ) Relative protein mass composition of the C. trachomatis EB. Approximately 2% of total EB protein mass is comprised of T3S chaperones, with Slc1, Scc2 and Mcsc accounting for over 99% of their mass. This figure represents a reanalysis of data reported in [31] . B ) Protein interaction network for Slc1 and Mcsc. Cell lysates from gradient purified EBs were incubated with anti-Slc1 antibodies, anti-Mcsc antibodies, or non-specific IgG cross-linked to an agarose resin. Bound proteins were eluted at low pH, digested with trypsin and identified by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) ( Table S1 ). Only proteins that displayed specific interactions are shown. C ) Specificity of Slc1-interactions. EB lysates were incubated with either anti-Slc1 antisera or pre-immune sera, and bound proteins were captured on Protein A/G agarose resin. The eluate (bound) and flow through (unbound) from both Slc1 IP and control IP were analyzed by immunoblotting with antibodies against selected proteins. MOMP is an abundant Chlamydia protein that serves as control for the specificity of the interactions shown.

Techniques Used: Purification, Incubation, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Flow Cytometry

44) Product Images from "Activity of a Bacterial Cell Envelope Stress Response Is Controlled by the Interaction of a Protein Binding Domain with Different Partners *"

Article Title: Activity of a Bacterial Cell Envelope Stress Response Is Controlled by the Interaction of a Protein Binding Domain with Different Partners *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.614107

Evidence that interaction of the PspC C-terminal domain with PspA or PspB is mutually exclusive in vitro . A , summary of the protocol used for the GST fusion protein two-phase membrane lysate pulldown assay. B , immunoblot analysis. In Experiment A , GST or GST fused to the PspC C-terminal domain ( GST-PspC CT ) was bound to glutathione-Sepharose (beads) and incubated with a detergent-solubilized membrane lysate from a Y. enterocolitica strain in which the only core Psp protein present was PspA ( prey lysate 1 ). After washing, proteins were recovered from half of the beads by boiling in SDS-PAGE sample buffer ( Elution 1 ). The other half of the beads was incubated with a second detergent-solubilized membrane lysate from a Y. enterocolitica strain in which the only core Psp protein present was PspB ( prey lysate 2 ). After washing, proteins were recovered by boiling in SDS-PAGE sample buffer ( Elution 2 ). Experiment B was done similarly, except that the order of incubation with the PspA and PspB membrane lysates was reversed. For each experiment, membrane lysates ( Inputs ) and recovered proteins ( Elutions ) were analyzed by SDS-PAGE and immunoblotting with PspA or PspB antiserum. The GST fusion protein in each elution was detected by Ponceau S staining of the immunoblot membrane (for experiments A and B the elution samples for Ponceau S staining were run on the same gels, but irrelevant lanes between elutions 1 and 2 have been removed).
Figure Legend Snippet: Evidence that interaction of the PspC C-terminal domain with PspA or PspB is mutually exclusive in vitro . A , summary of the protocol used for the GST fusion protein two-phase membrane lysate pulldown assay. B , immunoblot analysis. In Experiment A , GST or GST fused to the PspC C-terminal domain ( GST-PspC CT ) was bound to glutathione-Sepharose (beads) and incubated with a detergent-solubilized membrane lysate from a Y. enterocolitica strain in which the only core Psp protein present was PspA ( prey lysate 1 ). After washing, proteins were recovered from half of the beads by boiling in SDS-PAGE sample buffer ( Elution 1 ). The other half of the beads was incubated with a second detergent-solubilized membrane lysate from a Y. enterocolitica strain in which the only core Psp protein present was PspB ( prey lysate 2 ). After washing, proteins were recovered by boiling in SDS-PAGE sample buffer ( Elution 2 ). Experiment B was done similarly, except that the order of incubation with the PspA and PspB membrane lysates was reversed. For each experiment, membrane lysates ( Inputs ) and recovered proteins ( Elutions ) were analyzed by SDS-PAGE and immunoblotting with PspA or PspB antiserum. The GST fusion protein in each elution was detected by Ponceau S staining of the immunoblot membrane (for experiments A and B the elution samples for Ponceau S staining were run on the same gels, but irrelevant lanes between elutions 1 and 2 have been removed).

Techniques Used: In Vitro, Incubation, SDS Page, Staining

GST-PspC CT fusion protein pulldown assay. GST, GST fused to the PspC C-terminal domain ( GST-PspC CT ), or a derivative with the V125D mutation ( GST-PspC CT-V125D ) was bound to glutathione-Sepharose (beads) and incubated with a detergent-solubilized membrane lysate from a Y. enterocolitica strain with all core Psp proteins (Psp + ) or in which the only core Psp proteins present were PspA or PspB as indicated ( Prey ). After washing, proteins were recovered by boiling in SDS-PAGE sample buffer. Membrane lysates ( Inputs ) and recovered proteins ( Elutions ) were analyzed by SDS-PAGE and immunoblotting with PspA or PspB antiserum. The GST fusion protein in each elution was detected by Ponceau S staining of the immunoblot membrane.
Figure Legend Snippet: GST-PspC CT fusion protein pulldown assay. GST, GST fused to the PspC C-terminal domain ( GST-PspC CT ), or a derivative with the V125D mutation ( GST-PspC CT-V125D ) was bound to glutathione-Sepharose (beads) and incubated with a detergent-solubilized membrane lysate from a Y. enterocolitica strain with all core Psp proteins (Psp + ) or in which the only core Psp proteins present were PspA or PspB as indicated ( Prey ). After washing, proteins were recovered by boiling in SDS-PAGE sample buffer. Membrane lysates ( Inputs ) and recovered proteins ( Elutions ) were analyzed by SDS-PAGE and immunoblotting with PspA or PspB antiserum. The GST fusion protein in each elution was detected by Ponceau S staining of the immunoblot membrane.

Techniques Used: Mutagenesis, Incubation, SDS Page, Staining

In vitro GST/MBP fusion protein interaction assay. GST, GST fused to the PspC C-terminal domain ( GST-PspC CT ), or a derivative with the V125D mutation ( GST-PspC CT-V125D ) were bound to glutathione-Sepharose (beads) and incubated with 15 μg of MBP fused to the C-terminal domain of PspB ( MBP-PspB CT ) or to LacZα ( MBP-LacZ α). After washing, proteins were recovered by boiling in SDS-PAGE sample buffer. Samples of each purified MBP-fusion protein ( Inputs ) and the recovered proteins ( Elutions ) were analyzed by SDS-PAGE and immunoblotting with anti-MBP or anti-GST monoclonal antibodies. MBP-LacZα underwent apparent degradation during purification, leading to the isolation of both full-length and truncated protein.
Figure Legend Snippet: In vitro GST/MBP fusion protein interaction assay. GST, GST fused to the PspC C-terminal domain ( GST-PspC CT ), or a derivative with the V125D mutation ( GST-PspC CT-V125D ) were bound to glutathione-Sepharose (beads) and incubated with 15 μg of MBP fused to the C-terminal domain of PspB ( MBP-PspB CT ) or to LacZα ( MBP-LacZ α). After washing, proteins were recovered by boiling in SDS-PAGE sample buffer. Samples of each purified MBP-fusion protein ( Inputs ) and the recovered proteins ( Elutions ) were analyzed by SDS-PAGE and immunoblotting with anti-MBP or anti-GST monoclonal antibodies. MBP-LacZα underwent apparent degradation during purification, leading to the isolation of both full-length and truncated protein.

Techniques Used: In Vitro, Protein Interaction Assay, Mutagenesis, Incubation, SDS Page, Purification, Isolation

45) Product Images from "p66? and p66? of the Mi-2/NuRD complex mediate MBD2 and histone interaction"

Article Title: p66? and p66? of the Mi-2/NuRD complex mediate MBD2 and histone interaction

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkj437

Acetylation of histone tails reduces the association with both p66-proteins in vitro . ( A ) The histone acetyltransferase domains of p300 and of PCAF were bacterially expressed and incubated with individual GST-histone tails together with radioactive acetyl-CoA. Glutathione–Sepharose purified GST-histone tails were eluted with SDS sample buffer, fractionated on SDS–PAGE and visualized by fluorography. p300 acetylated primarily H2A, H3 and H4 histone tails, whereas PCAF acetylated primarily the tails of histone H3, and also of H4. Acetylation by p300 or PCAF reduced association of p66α ( B ) or of p66β ( C ) with acetylated histone tails. Acetylated GST-histone tails as in (A), but incubated with non-radioactive acetyl-CoA (+CoA) or non-acetylated histone tails (−CoA), were incubated with [ 35 S]methionine-labeled in vitro translated p66-proteins. All samples were purified by glutathione–Sepharose, fractionated on SDS–PAGE and visualized by autoradiography.
Figure Legend Snippet: Acetylation of histone tails reduces the association with both p66-proteins in vitro . ( A ) The histone acetyltransferase domains of p300 and of PCAF were bacterially expressed and incubated with individual GST-histone tails together with radioactive acetyl-CoA. Glutathione–Sepharose purified GST-histone tails were eluted with SDS sample buffer, fractionated on SDS–PAGE and visualized by fluorography. p300 acetylated primarily H2A, H3 and H4 histone tails, whereas PCAF acetylated primarily the tails of histone H3, and also of H4. Acetylation by p300 or PCAF reduced association of p66α ( B ) or of p66β ( C ) with acetylated histone tails. Acetylated GST-histone tails as in (A), but incubated with non-radioactive acetyl-CoA (+CoA) or non-acetylated histone tails (−CoA), were incubated with [ 35 S]methionine-labeled in vitro translated p66-proteins. All samples were purified by glutathione–Sepharose, fractionated on SDS–PAGE and visualized by autoradiography.

Techniques Used: In Vitro, Incubation, Purification, SDS Page, Labeling, Autoradiography

K149 of p66α is required for the MBD2 interaction as well as for the MBD2-mediated repression. ( A ) HEK293 cells were harvested 48 h after transfection with various combinations of DNA constructs, as indicated above the figure. Nuclear protein extracts were prepared (input) and purified with glutathione–Sepharose beads. The bound protein together with the input fractions were analyzed by western blotting using the anti-Gal antibody. ( B ) K149R mutant of p66α decreases MBD2-mediated repression. HeLa cells were cotransfected with a 4xUAStk luciferase reporter together with vectors expressing the Gal-DNA binding domain, or Gal-MBD2b and increasing amount of pSG5-p66α or pSG5-p66αK149R. Fold repression was determined relative to the Gal-DNA binding domain, significant changes relative to Gal-MBD2b (asterisk) and relative to comparable amounts of p66α (open triangle) are indicated.
Figure Legend Snippet: K149 of p66α is required for the MBD2 interaction as well as for the MBD2-mediated repression. ( A ) HEK293 cells were harvested 48 h after transfection with various combinations of DNA constructs, as indicated above the figure. Nuclear protein extracts were prepared (input) and purified with glutathione–Sepharose beads. The bound protein together with the input fractions were analyzed by western blotting using the anti-Gal antibody. ( B ) K149R mutant of p66α decreases MBD2-mediated repression. HeLa cells were cotransfected with a 4xUAStk luciferase reporter together with vectors expressing the Gal-DNA binding domain, or Gal-MBD2b and increasing amount of pSG5-p66α or pSG5-p66αK149R. Fold repression was determined relative to the Gal-DNA binding domain, significant changes relative to Gal-MBD2b (asterisk) and relative to comparable amounts of p66α (open triangle) are indicated.

Techniques Used: Transfection, Construct, Purification, Western Blot, Mutagenesis, Luciferase, Expressing, Binding Assay

Both p66α and p66β interact with histone tails in vitro . GST and GST-histone tails were purified with glutathione–Sepharose beads and analyzed by SDS–PAGE to normalize protein amounts. Equivalent amounts of GST fusion proteins were incubated with [ 35 S]methionine-labeled in vitro translated p66-proteins, as indicated left of the figure. The bound proteins were eluted with SDS sample buffer, fractionated on SDS–PAGE and visualized by fluorography.
Figure Legend Snippet: Both p66α and p66β interact with histone tails in vitro . GST and GST-histone tails were purified with glutathione–Sepharose beads and analyzed by SDS–PAGE to normalize protein amounts. Equivalent amounts of GST fusion proteins were incubated with [ 35 S]methionine-labeled in vitro translated p66-proteins, as indicated left of the figure. The bound proteins were eluted with SDS sample buffer, fractionated on SDS–PAGE and visualized by fluorography.

Techniques Used: In Vitro, Purification, SDS Page, Incubation, Labeling

46) Product Images from "p66? and p66? of the Mi-2/NuRD complex mediate MBD2 and histone interaction"

Article Title: p66? and p66? of the Mi-2/NuRD complex mediate MBD2 and histone interaction

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkj437

Acetylation of histone tails reduces the association with both p66-proteins in vitro . ( A ) The histone acetyltransferase domains of p300 and of PCAF were bacterially expressed and incubated with individual GST-histone tails together with radioactive acetyl-CoA. Glutathione–Sepharose purified GST-histone tails were eluted with SDS sample buffer, fractionated on SDS–PAGE and visualized by fluorography. p300 acetylated primarily H2A, H3 and H4 histone tails, whereas PCAF acetylated primarily the tails of histone H3, and also of H4. Acetylation by p300 or PCAF reduced association of p66α ( B ) or of p66β ( C ) with acetylated histone tails. Acetylated GST-histone tails as in (A), but incubated with non-radioactive acetyl-CoA (+CoA) or non-acetylated histone tails (−CoA), were incubated with [ 35 S]methionine-labeled in vitro translated p66-proteins. All samples were purified by glutathione–Sepharose, fractionated on SDS–PAGE and visualized by autoradiography.
Figure Legend Snippet: Acetylation of histone tails reduces the association with both p66-proteins in vitro . ( A ) The histone acetyltransferase domains of p300 and of PCAF were bacterially expressed and incubated with individual GST-histone tails together with radioactive acetyl-CoA. Glutathione–Sepharose purified GST-histone tails were eluted with SDS sample buffer, fractionated on SDS–PAGE and visualized by fluorography. p300 acetylated primarily H2A, H3 and H4 histone tails, whereas PCAF acetylated primarily the tails of histone H3, and also of H4. Acetylation by p300 or PCAF reduced association of p66α ( B ) or of p66β ( C ) with acetylated histone tails. Acetylated GST-histone tails as in (A), but incubated with non-radioactive acetyl-CoA (+CoA) or non-acetylated histone tails (−CoA), were incubated with [ 35 S]methionine-labeled in vitro translated p66-proteins. All samples were purified by glutathione–Sepharose, fractionated on SDS–PAGE and visualized by autoradiography.

Techniques Used: In Vitro, Incubation, Purification, SDS Page, Labeling, Autoradiography

K149 of p66α is required for the MBD2 interaction as well as for the MBD2-mediated repression. ( A ) HEK293 cells were harvested 48 h after transfection with various combinations of DNA constructs, as indicated above the figure. Nuclear protein extracts were prepared (input) and purified with glutathione–Sepharose beads. The bound protein together with the input fractions were analyzed by western blotting using the anti-Gal antibody. ( B ) K149R mutant of p66α decreases MBD2-mediated repression. HeLa cells were cotransfected with a 4xUAStk luciferase reporter together with vectors expressing the Gal-DNA binding domain, or Gal-MBD2b and increasing amount of pSG5-p66α or pSG5-p66αK149R. Fold repression was determined relative to the Gal-DNA binding domain, significant changes relative to Gal-MBD2b (asterisk) and relative to comparable amounts of p66α (open triangle) are indicated.
Figure Legend Snippet: K149 of p66α is required for the MBD2 interaction as well as for the MBD2-mediated repression. ( A ) HEK293 cells were harvested 48 h after transfection with various combinations of DNA constructs, as indicated above the figure. Nuclear protein extracts were prepared (input) and purified with glutathione–Sepharose beads. The bound protein together with the input fractions were analyzed by western blotting using the anti-Gal antibody. ( B ) K149R mutant of p66α decreases MBD2-mediated repression. HeLa cells were cotransfected with a 4xUAStk luciferase reporter together with vectors expressing the Gal-DNA binding domain, or Gal-MBD2b and increasing amount of pSG5-p66α or pSG5-p66αK149R. Fold repression was determined relative to the Gal-DNA binding domain, significant changes relative to Gal-MBD2b (asterisk) and relative to comparable amounts of p66α (open triangle) are indicated.

Techniques Used: Transfection, Construct, Purification, Western Blot, Mutagenesis, Luciferase, Expressing, Binding Assay

Both p66α and p66β interact with histone tails in vitro . GST and GST-histone tails were purified with glutathione–Sepharose beads and analyzed by SDS–PAGE to normalize protein amounts. Equivalent amounts of GST fusion proteins were incubated with [ 35 S]methionine-labeled in vitro translated p66-proteins, as indicated left of the figure. The bound proteins were eluted with SDS sample buffer, fractionated on SDS–PAGE and visualized by fluorography.
Figure Legend Snippet: Both p66α and p66β interact with histone tails in vitro . GST and GST-histone tails were purified with glutathione–Sepharose beads and analyzed by SDS–PAGE to normalize protein amounts. Equivalent amounts of GST fusion proteins were incubated with [ 35 S]methionine-labeled in vitro translated p66-proteins, as indicated left of the figure. The bound proteins were eluted with SDS sample buffer, fractionated on SDS–PAGE and visualized by fluorography.

Techniques Used: In Vitro, Purification, SDS Page, Incubation, Labeling

47) Product Images from "Fragile Mental Retardation Protein Interacts with the RNA-Binding Protein Caprin1 in Neuronal RiboNucleoProtein Complexes"

Article Title: Fragile Mental Retardation Protein Interacts with the RNA-Binding Protein Caprin1 in Neuronal RiboNucleoProtein Complexes

Journal: PLoS ONE

doi: 10.1371/journal.pone.0039338

mRNAs associated with Caprin1 and FMRP. A ) mRNAs co-immunoprecipitated by mAb7G1-1 and IgY#C10 from WT and KO brain extracts were analyzed by RT-PCR and visualized by agarose gels (left panels) and by Light Cycler RT-PCR (right panels). Bars in red refer to mRNA targets to Caprin1, in blue common to Caprin1 and FMRP, in green to FMRP and in black to non-targets mRNAs. Dark colors refer to WT and pale to KO, respectively. N = 5, P≤0.001 of a pool of 10 adult brains. Vertical black dot lines represent thresholds corresponding to background. B ) RT-PCR analyses of selected mRNAs co-immunoprecipitated with Caprin1.
Figure Legend Snippet: mRNAs associated with Caprin1 and FMRP. A ) mRNAs co-immunoprecipitated by mAb7G1-1 and IgY#C10 from WT and KO brain extracts were analyzed by RT-PCR and visualized by agarose gels (left panels) and by Light Cycler RT-PCR (right panels). Bars in red refer to mRNA targets to Caprin1, in blue common to Caprin1 and FMRP, in green to FMRP and in black to non-targets mRNAs. Dark colors refer to WT and pale to KO, respectively. N = 5, P≤0.001 of a pool of 10 adult brains. Vertical black dot lines represent thresholds corresponding to background. B ) RT-PCR analyses of selected mRNAs co-immunoprecipitated with Caprin1.

Techniques Used: Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

mAb7G1-1 detects mFMRP and Caprin1. A ) Immunoprecipitation analyses of WT and KO2 mouse brain extracts with mAb7G1-1 followed by immunoblotting with mAb1C3 (left panel in A ) or with mAb7G1-1 (right panel in A ). In addition to mFMRP, a clear band at 116 kDa is detected in WT immunoprecipitates. A similar band is also detected in KO2 extracts. Both bands are absent when immunoprecipitation is performed in the presence of the epitope peptide KHLDTKENTHFSQPN. B ) Immunoblot analyses with mAb7G1-1 of 3T3, STEK and HeLa cell extracts. While mAb3C1 detects only mFMRP in 3T3 extracts, mAb7G1-1 reacts with both mFMRP and p116. An additional weak band is also detected in both extracts at 65 kDa (indicated by a star). mAb7G1-1 does not react with hFMRP from HeLa extracts, but recognizes p116. Note the presence of the additional band that migrates slightly above 65 kDa in human HeLa extracts (double star). C ) Extracts from STEK cells were immunoprecipitated with mAb7G1-1. Immunoblot analyses with mAb1C3 reveal that mFMRP is indeed absent, while mAb7G1-1 reacts with p116. The Coomassie brillant blue stained band at 116 kDa was excised and analyzed by mass spectrometry and was identified as Caprin1. Immunoblot analyses with rabbit antisera raised against hCaprin1 confirmed the nature of p116 as Caprin1. D ) In vitro translated 35 S-labeled Caprin1 is immunoprecipitated by mAb7G1-1. E ) Recombinant GST-Caprin1 isolated on Glutathione-Sepharose beads is revealed with anti-Caprin1 IgG in immunoblot analyses. Note the presence of the minor truncated band at ∼95 kDa. F ) Structural comparisons between FMRP and Caprin1. WT and KO: wild type C57BL/6J and Fmr1 −/− KO2 mice, respectively. IP: immunoprecipitation; IB: immunoblot; Cont: control; HC: IgG heavy chains; AP: affinity purified; AD: affinity depleted; AR: autoradiography.
Figure Legend Snippet: mAb7G1-1 detects mFMRP and Caprin1. A ) Immunoprecipitation analyses of WT and KO2 mouse brain extracts with mAb7G1-1 followed by immunoblotting with mAb1C3 (left panel in A ) or with mAb7G1-1 (right panel in A ). In addition to mFMRP, a clear band at 116 kDa is detected in WT immunoprecipitates. A similar band is also detected in KO2 extracts. Both bands are absent when immunoprecipitation is performed in the presence of the epitope peptide KHLDTKENTHFSQPN. B ) Immunoblot analyses with mAb7G1-1 of 3T3, STEK and HeLa cell extracts. While mAb3C1 detects only mFMRP in 3T3 extracts, mAb7G1-1 reacts with both mFMRP and p116. An additional weak band is also detected in both extracts at 65 kDa (indicated by a star). mAb7G1-1 does not react with hFMRP from HeLa extracts, but recognizes p116. Note the presence of the additional band that migrates slightly above 65 kDa in human HeLa extracts (double star). C ) Extracts from STEK cells were immunoprecipitated with mAb7G1-1. Immunoblot analyses with mAb1C3 reveal that mFMRP is indeed absent, while mAb7G1-1 reacts with p116. The Coomassie brillant blue stained band at 116 kDa was excised and analyzed by mass spectrometry and was identified as Caprin1. Immunoblot analyses with rabbit antisera raised against hCaprin1 confirmed the nature of p116 as Caprin1. D ) In vitro translated 35 S-labeled Caprin1 is immunoprecipitated by mAb7G1-1. E ) Recombinant GST-Caprin1 isolated on Glutathione-Sepharose beads is revealed with anti-Caprin1 IgG in immunoblot analyses. Note the presence of the minor truncated band at ∼95 kDa. F ) Structural comparisons between FMRP and Caprin1. WT and KO: wild type C57BL/6J and Fmr1 −/− KO2 mice, respectively. IP: immunoprecipitation; IB: immunoblot; Cont: control; HC: IgG heavy chains; AP: affinity purified; AD: affinity depleted; AR: autoradiography.

Techniques Used: Immunoprecipitation, Staining, Mass Spectrometry, In Vitro, Labeling, Recombinant, Isolation, Mouse Assay, Affinity Purification, Autoradiography

48) Product Images from "Promiscuous interaction of SNAP-25 with all plasma membrane syntaxins in a neuroendocrine cell"

Article Title: Promiscuous interaction of SNAP-25 with all plasma membrane syntaxins in a neuroendocrine cell

Journal: Biochemical Journal

doi: 10.1042/BJ20050583

Syntaxins 3 and 4 are present in the brain, and are capable of in vitro interaction with both SNAP-25 and synaptobrevin 2 ( A ) Western immunoblot analysis of rat internal organs and different parts of brain. Protein normalization (30 μg per lane) was verified by Coomassie staining (CS). ( B ) Recombinant syntaxins 3 and 4 form SDS-resistant SNARE complexes upon addition of SNAP-25 and synaptobrevin 2 (syb2) in a 30 min reaction at 24 °C. Coomassie stained gel. ( C ) Syntaxins 3 and 4 (Syx3 and Syx4) are as capable of direct binding to SNAP-25 as syntaxin 1. Immobilized GST fusion proteins of syntaxins were incubated with SNAP-25 for 30 min and, after extensive washing, were analysed by SDS/PAGE and Sypro Orange staining. A control reaction was performed using glutathione–Sepharose beads without GST–syntaxin, but in the presence of SNAP-25.
Figure Legend Snippet: Syntaxins 3 and 4 are present in the brain, and are capable of in vitro interaction with both SNAP-25 and synaptobrevin 2 ( A ) Western immunoblot analysis of rat internal organs and different parts of brain. Protein normalization (30 μg per lane) was verified by Coomassie staining (CS). ( B ) Recombinant syntaxins 3 and 4 form SDS-resistant SNARE complexes upon addition of SNAP-25 and synaptobrevin 2 (syb2) in a 30 min reaction at 24 °C. Coomassie stained gel. ( C ) Syntaxins 3 and 4 (Syx3 and Syx4) are as capable of direct binding to SNAP-25 as syntaxin 1. Immobilized GST fusion proteins of syntaxins were incubated with SNAP-25 for 30 min and, after extensive washing, were analysed by SDS/PAGE and Sypro Orange staining. A control reaction was performed using glutathione–Sepharose beads without GST–syntaxin, but in the presence of SNAP-25.

Techniques Used: In Vitro, Western Blot, Staining, Recombinant, Binding Assay, Incubation, SDS Page

SNAP-25 interacts in vivo with all four plasma membrane syntaxin isoforms ( A ) Immunoprecipitation experiments of PC12 cell extracts using either control Sepharose beads or Sepharose beads with a covalently attached monoclonal anti-SNAP-25 antibody are shown. A fraction of the load (one-hundredth) and of the pull-down material (one-tenth) was probed using rabbit antibodies against the indicated proteins. ( B ) MS analysis of the anti-SNAP-25 pull-down material reveals partial peptide coverage for all four plasma membrane syntaxin isoforms.
Figure Legend Snippet: SNAP-25 interacts in vivo with all four plasma membrane syntaxin isoforms ( A ) Immunoprecipitation experiments of PC12 cell extracts using either control Sepharose beads or Sepharose beads with a covalently attached monoclonal anti-SNAP-25 antibody are shown. A fraction of the load (one-hundredth) and of the pull-down material (one-tenth) was probed using rabbit antibodies against the indicated proteins. ( B ) MS analysis of the anti-SNAP-25 pull-down material reveals partial peptide coverage for all four plasma membrane syntaxin isoforms.

Techniques Used: In Vivo, Immunoprecipitation, Mass Spectrometry

49) Product Images from "Proteomic analysis of tyrosine phosphorylation during human liver transplantation"

Article Title: Proteomic analysis of tyrosine phosphorylation during human liver transplantation

Journal: Proteome Science

doi: 10.1186/1477-5956-5-1

Identification of tyrosine phosphorylated proteins upon I/R . A . Schematic representation of the approach used for tyrosine phosphorylated proteins identification. PY matrix: anti-phosphotyrosine antibodies coupled to agarose beads. B . Representative SDS-PAGE experiment after tyrosine immunoprecipitation (Ip PY) of 60 min ischemia (I60) and 60 min reperfusion (R60) protein extracts. The band marked by an arrow has been further identified as the SH 2 /SH 3 adaptor Nck-1. N = 1 on 2 independent pools of 3 liver biopsy protein extracts. C . Pie-chart representation of the total number of proteins identified with a significant Mascot score by at least one unique peptide for the 60 min ischemia (I60, left panel) and the 60 min reperfusion (R60, right panel). The proteins were classified in functional families according to their GO (Gene Ontology) annotation.
Figure Legend Snippet: Identification of tyrosine phosphorylated proteins upon I/R . A . Schematic representation of the approach used for tyrosine phosphorylated proteins identification. PY matrix: anti-phosphotyrosine antibodies coupled to agarose beads. B . Representative SDS-PAGE experiment after tyrosine immunoprecipitation (Ip PY) of 60 min ischemia (I60) and 60 min reperfusion (R60) protein extracts. The band marked by an arrow has been further identified as the SH 2 /SH 3 adaptor Nck-1. N = 1 on 2 independent pools of 3 liver biopsy protein extracts. C . Pie-chart representation of the total number of proteins identified with a significant Mascot score by at least one unique peptide for the 60 min ischemia (I60, left panel) and the 60 min reperfusion (R60, right panel). The proteins were classified in functional families according to their GO (Gene Ontology) annotation.

Techniques Used: SDS Page, Immunoprecipitation, Functional Assay

Determination of Nck-1 interactants by GST pull-down . A . Schematic representation of the approach used for tyrosine mass spectrometry identification of GST Nck-1 binding proteins. GST Nck-1 matrix: GST Nck-1 fusion protein coupled to sepharose beads. B . Representative SDS-PAGE experiment after GST Nck-1 pull-down of 0 (I0) and 60 min ischemia (I60) and 0 (R0) and 60 min reperfusion (R60) protein extract. Bands corresponding to GST Nck-1 fusion protein and cleavage products (Nck-1 and GST) are marked by arrows. N = 1 on 2 independent pools of 3 liver biopsy protein extracts. C . Venn diagram representation of the proteins identified after mass spectrometry analysis of GST Nck-1 binding proteins.
Figure Legend Snippet: Determination of Nck-1 interactants by GST pull-down . A . Schematic representation of the approach used for tyrosine mass spectrometry identification of GST Nck-1 binding proteins. GST Nck-1 matrix: GST Nck-1 fusion protein coupled to sepharose beads. B . Representative SDS-PAGE experiment after GST Nck-1 pull-down of 0 (I0) and 60 min ischemia (I60) and 0 (R0) and 60 min reperfusion (R60) protein extract. Bands corresponding to GST Nck-1 fusion protein and cleavage products (Nck-1 and GST) are marked by arrows. N = 1 on 2 independent pools of 3 liver biopsy protein extracts. C . Venn diagram representation of the proteins identified after mass spectrometry analysis of GST Nck-1 binding proteins.

Techniques Used: Mass Spectrometry, Binding Assay, SDS Page

50) Product Images from "A new class of cyclin dependent kinase in Chlamydomonas is required for coupling cell size to cell division"

Article Title: A new class of cyclin dependent kinase in Chlamydomonas is required for coupling cell size to cell division

Journal: eLife

doi: 10.7554/eLife.10767

Cell cycle progression and complementation of cdkg1-2 . ( A ) Schematic representation of the CDKG1 locus and cdkg1-2 allele caused by insertion of a NIT1 plasmid and accompanying deletion (upper bracketed region). Coordinates are from genome assembly V5.5 taken from Phytozome ( http://phytozome.jgi.doe.gov ). Tall black rectangles represent exons and medium rectangles represent untranslated regions (UTR). Thin lines represent introns and intergenic regions. The lower bracketed region marks the 1.7kb 3’ UTR of CDKG1. Arrows represent the transcriptional starts and direction of transcription for URH1 and CDKG1 . Arrowheads mark binding sites for PCR primers used for genotyping: a1/b1 for CDKG1 last exon and adjacent 3’ UTR region, a4/b1 for NIT1 and CDKG1 3’ UTR junction (see Supplementary file 2 for detailed information). The 7 kb genomic region of the CDKG1 locus that was used to generate the HA-gCDKG1 complementation construct is shown below. The orange rectangle indicates the 3xHA tag located just downstream of the start codon. Primer sets a2/b2 and a3/b2 were used in RT-PCR experiments to amplify only HA-CDKG1 (a2/b2) or both endogenous and HA-CDKG1 cDNAs (a3/b2). ( B ) Ethidium bromide stained agarose gel showing genotyping PCR results for indicated strains. wt , wild-type; cdkg1-2, CDKG1 mutant strain; wt:HA-gCDKG1, transgenic line expressing HA-gCDKG1 ;. Results from representative non-complemented (1) and complemented (2, 3) progeny from a cross between cdkg1-2 and wt::HA-gCDKG1 are shown. Gene specific primer sets are shown on the right. Size phenotypes (large or wild type (WT)) are shown below. * Primer dimer. ( C ) Ethidium bromide stained agarose gel of RT-PCR products showing expression of HA-CDKG1 in a complemented cdkg1-2::HA-gCDKG1 strain. Strain genotypes are as described in panel ( B ). Gene specific amplicons and primer sets described in panel ( A ) are indicated to the right of each gel image. GBLP is an internal control. ( D ) Quantitative RT-PCR showing CDKG1 mRNA levels in synchronized wild-type ( WT ) or cdkg1-2::HA-gCDKG1 strains. All data were normalized to expression of internal control gene GBLP . Relative expression levels are shown based on values at time 0 hr that were set to 1. Error bars: S.D. of three replicates. ( E ) 12 hr light/12 hr dark synchronized wild type (black lines) and cdkg1-2 (orange lines) strains were monitored for passage through commitment (dashed lines), and for mitotic index (percentage in S/M phases) by light microscopy with fixed samples. The median cell size and time when the cultures had ~60% Committed cells is indicated by the dashed lines. DOI: http://dx.doi.org/10.7554/eLife.10767.004
Figure Legend Snippet: Cell cycle progression and complementation of cdkg1-2 . ( A ) Schematic representation of the CDKG1 locus and cdkg1-2 allele caused by insertion of a NIT1 plasmid and accompanying deletion (upper bracketed region). Coordinates are from genome assembly V5.5 taken from Phytozome ( http://phytozome.jgi.doe.gov ). Tall black rectangles represent exons and medium rectangles represent untranslated regions (UTR). Thin lines represent introns and intergenic regions. The lower bracketed region marks the 1.7kb 3’ UTR of CDKG1. Arrows represent the transcriptional starts and direction of transcription for URH1 and CDKG1 . Arrowheads mark binding sites for PCR primers used for genotyping: a1/b1 for CDKG1 last exon and adjacent 3’ UTR region, a4/b1 for NIT1 and CDKG1 3’ UTR junction (see Supplementary file 2 for detailed information). The 7 kb genomic region of the CDKG1 locus that was used to generate the HA-gCDKG1 complementation construct is shown below. The orange rectangle indicates the 3xHA tag located just downstream of the start codon. Primer sets a2/b2 and a3/b2 were used in RT-PCR experiments to amplify only HA-CDKG1 (a2/b2) or both endogenous and HA-CDKG1 cDNAs (a3/b2). ( B ) Ethidium bromide stained agarose gel showing genotyping PCR results for indicated strains. wt , wild-type; cdkg1-2, CDKG1 mutant strain; wt:HA-gCDKG1, transgenic line expressing HA-gCDKG1 ;. Results from representative non-complemented (1) and complemented (2, 3) progeny from a cross between cdkg1-2 and wt::HA-gCDKG1 are shown. Gene specific primer sets are shown on the right. Size phenotypes (large or wild type (WT)) are shown below. * Primer dimer. ( C ) Ethidium bromide stained agarose gel of RT-PCR products showing expression of HA-CDKG1 in a complemented cdkg1-2::HA-gCDKG1 strain. Strain genotypes are as described in panel ( B ). Gene specific amplicons and primer sets described in panel ( A ) are indicated to the right of each gel image. GBLP is an internal control. ( D ) Quantitative RT-PCR showing CDKG1 mRNA levels in synchronized wild-type ( WT ) or cdkg1-2::HA-gCDKG1 strains. All data were normalized to expression of internal control gene GBLP . Relative expression levels are shown based on values at time 0 hr that were set to 1. Error bars: S.D. of three replicates. ( E ) 12 hr light/12 hr dark synchronized wild type (black lines) and cdkg1-2 (orange lines) strains were monitored for passage through commitment (dashed lines), and for mitotic index (percentage in S/M phases) by light microscopy with fixed samples. The median cell size and time when the cultures had ~60% Committed cells is indicated by the dashed lines. DOI: http://dx.doi.org/10.7554/eLife.10767.004

Techniques Used: Plasmid Preparation, Binding Assay, Polymerase Chain Reaction, Construct, Reverse Transcription Polymerase Chain Reaction, Staining, Agarose Gel Electrophoresis, Mutagenesis, Transgenic Assay, Expressing, Quantitative RT-PCR, Light Microscopy

Expression profiles and interactions between D cyclins, CDKG1 and MAT3/RBR during the cell cycle. ( A ) Ethidium bromide stained agarose gels showing amplification of cDNAs made with RNA samples taken from synchronized cultures in a 14 hr light/10 hr dark cycle at four stages: daughter cells (1 hr light), post-commitment cells (10 hr light), S/M phase cells (1 hr dark) and post-mitotic cells (4 hr dark). Primers used are listed in Supplementary file 2 and the number of PCR amplification cycles used for each reaction is displayed on the right. GBLP is an internal control. ( B ) Profiles of CDKG1, CDKB1 and GBLP mRNAs as described in ( A ), except the pre-commitment sample was from 4 hr light. ( C ) Interaction of CYCD3 AxAxA mutant with CDKG1 and MAT3. Mutated CYCD3 (LxCxE→AxAxA) was fused to the Gal4 activation domain (AD) and tested in an Y2H assay with MAT3/RBR and CDKG1. Empty indicates vector-only with no fusion protein. Growth of two independent co-transformants is shown, and the relative strength of interaction is indicated by -, no interaction, +, weak interaction, and +++, very strong interaction. ( D ) Bar graph shows relative kinase activity of IVT CDKG1, CDKG1 kd and CDKG1+CYCD3 using GST-MAT3 as a substrate. The amount of 32 P-labeled GST-MAT3 from each reaction was normalized to the value from lane 2. Data are expressed as the mean of three independent experiments. Error bar: S.D. ( E ) Immunoprecipitation (IP) from whole cell extracts, fractionation by SDS-PAGE and detection of CDKG1 by Western blotting using polyclonal antisera raised against full length CDGK1. All strains used were synchronized in S/M phase. Lanes 1,2 are concentrated input fractions prior to IP from indicated strains. Anti-HA (lanes 3,4) or anti-CDKG1 (lanes 5–8) antibodies were used for IPs as indicated and the supernatant (lanes 3,5,7) or pellet (lanes 4,6,8) fractions probed using anti-CDKG1. The anti-CDKG1 antibody detects a single band near the predicted molecular weight of 44 kDa that is not present in cdkg1-2 mutants. ( F ) Schematic of the regulatory hierarchy for the Chlamydomonas mitotic sizer pathway (left side) and the metazoan G1 control pathway (right side). Both pathways integrate internal and/or external signals for cell cycle progression through D-cyclin dependent CDKs that phosphorylate RB-related proteins. DOI: http://dx.doi.org/10.7554/eLife.10767.009
Figure Legend Snippet: Expression profiles and interactions between D cyclins, CDKG1 and MAT3/RBR during the cell cycle. ( A ) Ethidium bromide stained agarose gels showing amplification of cDNAs made with RNA samples taken from synchronized cultures in a 14 hr light/10 hr dark cycle at four stages: daughter cells (1 hr light), post-commitment cells (10 hr light), S/M phase cells (1 hr dark) and post-mitotic cells (4 hr dark). Primers used are listed in Supplementary file 2 and the number of PCR amplification cycles used for each reaction is displayed on the right. GBLP is an internal control. ( B ) Profiles of CDKG1, CDKB1 and GBLP mRNAs as described in ( A ), except the pre-commitment sample was from 4 hr light. ( C ) Interaction of CYCD3 AxAxA mutant with CDKG1 and MAT3. Mutated CYCD3 (LxCxE→AxAxA) was fused to the Gal4 activation domain (AD) and tested in an Y2H assay with MAT3/RBR and CDKG1. Empty indicates vector-only with no fusion protein. Growth of two independent co-transformants is shown, and the relative strength of interaction is indicated by -, no interaction, +, weak interaction, and +++, very strong interaction. ( D ) Bar graph shows relative kinase activity of IVT CDKG1, CDKG1 kd and CDKG1+CYCD3 using GST-MAT3 as a substrate. The amount of 32 P-labeled GST-MAT3 from each reaction was normalized to the value from lane 2. Data are expressed as the mean of three independent experiments. Error bar: S.D. ( E ) Immunoprecipitation (IP) from whole cell extracts, fractionation by SDS-PAGE and detection of CDKG1 by Western blotting using polyclonal antisera raised against full length CDGK1. All strains used were synchronized in S/M phase. Lanes 1,2 are concentrated input fractions prior to IP from indicated strains. Anti-HA (lanes 3,4) or anti-CDKG1 (lanes 5–8) antibodies were used for IPs as indicated and the supernatant (lanes 3,5,7) or pellet (lanes 4,6,8) fractions probed using anti-CDKG1. The anti-CDKG1 antibody detects a single band near the predicted molecular weight of 44 kDa that is not present in cdkg1-2 mutants. ( F ) Schematic of the regulatory hierarchy for the Chlamydomonas mitotic sizer pathway (left side) and the metazoan G1 control pathway (right side). Both pathways integrate internal and/or external signals for cell cycle progression through D-cyclin dependent CDKs that phosphorylate RB-related proteins. DOI: http://dx.doi.org/10.7554/eLife.10767.009

Techniques Used: Expressing, Staining, Amplification, Polymerase Chain Reaction, Mutagenesis, Activation Assay, Y2H Assay, Plasmid Preparation, Activity Assay, Labeling, Immunoprecipitation, Fractionation, SDS Page, Western Blot, Molecular Weight

51) Product Images from "Structural and biochemical studies of SLIP1-SLBP identify DBP5 and eIF3g as SLIP1-binding proteins"

Article Title: Structural and biochemical studies of SLIP1-SLBP identify DBP5 and eIF3g as SLIP1-binding proteins

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkt558

Determinants and conservation of the SLIP1–SLBP interaction. ( A ) Close-up view of the interactions between SLIP1 (in orange) and the SBM of SLBP (in blue). The molecules are in a similar orientation as in Figure 1 A. Selected residues are shown in stick representation and labeled. ( B ) Alignment of SLIP1 orthologues from Homo sapiens ( H.s. , Uniprot accession code A9UHW6), Xenopus laevis ( X.l. , Uniprot accession code Q801N6) and Danio rerio (D.r., Uniprot accession code Q5EAQ1). The secondary-structure elements observed in the D.r. SLIP1–SLBP 89–105 crystal structure are shown below the sequences (h = helix). The HEAT repeats are labeled according to a superposition with MIF4G structure (PDB code 2VSO). Also labeled are the antiparallel helices of the MIF4G-like fold of SLIP1 (α1–α10). Highlighted in orange are conserved residues. The residues of SLIP1 interacting with the corresponding motif of SLBP are highlighted above the sequences (with M, in blue). The residues involved in dimerization are also highlighted (with D, in orange). The alignment also includes the C-terminal portion of the related human protein CTIF (Uniprot accession code O43310). ( C ) Alignment of the SBM of SLBP orthologues ( H.s. , Uniprot accession code Q14493; X.l. , Uniprot accession code P79943; D.r. , Uniprot accession code Q7SXI8) is shown with a similar representation as in panel B. The residues of SLBP involved in the interaction with SLIP1 are highlighted above the sequences (with S, in orange). ( D ) SLIP1 (lanes 1–4) or SLBP (lanes 5–8) was labeled with 35 S-methionine by in vitro translation in rabbit reticulocyte lysate. The lysates were incubated with GST, GST–SLIP1 or GST–SLIP1m (a mutant with the R77A/N81A/Q84A substitutions at the SBM-binding site). The complexes were bound to glutathione sepharose and analyzed by SDS gel electrophoresis and the bound proteins detected by autoradiography.
Figure Legend Snippet: Determinants and conservation of the SLIP1–SLBP interaction. ( A ) Close-up view of the interactions between SLIP1 (in orange) and the SBM of SLBP (in blue). The molecules are in a similar orientation as in Figure 1 A. Selected residues are shown in stick representation and labeled. ( B ) Alignment of SLIP1 orthologues from Homo sapiens ( H.s. , Uniprot accession code A9UHW6), Xenopus laevis ( X.l. , Uniprot accession code Q801N6) and Danio rerio (D.r., Uniprot accession code Q5EAQ1). The secondary-structure elements observed in the D.r. SLIP1–SLBP 89–105 crystal structure are shown below the sequences (h = helix). The HEAT repeats are labeled according to a superposition with MIF4G structure (PDB code 2VSO). Also labeled are the antiparallel helices of the MIF4G-like fold of SLIP1 (α1–α10). Highlighted in orange are conserved residues. The residues of SLIP1 interacting with the corresponding motif of SLBP are highlighted above the sequences (with M, in blue). The residues involved in dimerization are also highlighted (with D, in orange). The alignment also includes the C-terminal portion of the related human protein CTIF (Uniprot accession code O43310). ( C ) Alignment of the SBM of SLBP orthologues ( H.s. , Uniprot accession code Q14493; X.l. , Uniprot accession code P79943; D.r. , Uniprot accession code Q7SXI8) is shown with a similar representation as in panel B. The residues of SLBP involved in the interaction with SLIP1 are highlighted above the sequences (with S, in orange). ( D ) SLIP1 (lanes 1–4) or SLBP (lanes 5–8) was labeled with 35 S-methionine by in vitro translation in rabbit reticulocyte lysate. The lysates were incubated with GST, GST–SLIP1 or GST–SLIP1m (a mutant with the R77A/N81A/Q84A substitutions at the SBM-binding site). The complexes were bound to glutathione sepharose and analyzed by SDS gel electrophoresis and the bound proteins detected by autoradiography.

Techniques Used: Labeling, In Vitro, Incubation, Mutagenesis, Binding Assay, SDS-Gel, Electrophoresis, Autoradiography

Structure of SLIP1 bound to the SBM of DBP5. ( A ) Close-up view of the interactions between SLIP1 (in yellow) and the SBM of DBP5 (in violet). The molecules are in a similar orientation as in Figure 2 A. Selected residues are shown in stick representation and labeled. ( B ) Protein co-precipitations by GST pull-down assays. GST-tagged human DBP5 wild type or mutants were incubated with SLIP1 in a buffer containing 100 mM NaCl. One-sixth of the sample was kept as input control (upper panel), and the rest was co-precipitated with glutathione sepharose beads (lower panel). Both input and pull-down samples were analyzed on Coomassie stained 15% SDS PAGE. The lane on the left shows a molecular weight marker.
Figure Legend Snippet: Structure of SLIP1 bound to the SBM of DBP5. ( A ) Close-up view of the interactions between SLIP1 (in yellow) and the SBM of DBP5 (in violet). The molecules are in a similar orientation as in Figure 2 A. Selected residues are shown in stick representation and labeled. ( B ) Protein co-precipitations by GST pull-down assays. GST-tagged human DBP5 wild type or mutants were incubated with SLIP1 in a buffer containing 100 mM NaCl. One-sixth of the sample was kept as input control (upper panel), and the rest was co-precipitated with glutathione sepharose beads (lower panel). Both input and pull-down samples were analyzed on Coomassie stained 15% SDS PAGE. The lane on the left shows a molecular weight marker.

Techniques Used: Labeling, Incubation, Staining, SDS Page, Molecular Weight, Marker

52) Product Images from "An effector of Ypt6p binds the SNARE Tlg1p and mediates selective fusion of vesicles with late Golgi membranes"

Article Title: An effector of Ypt6p binds the SNARE Tlg1p and mediates selective fusion of vesicles with late Golgi membranes

Journal: The EMBO Journal

doi: 10.1093/emboj/20.21.5991

Fig. 3. The VFT complex binds directly to Ypt6p. ( A ) Purification of the VFT complex from yeast cells expressing Vps54p-PtA (strain SSY14). The complex was eluted from IgG–Sepharose with TEV protease, which cleaved off the PtA. Besides the protease (TEV), the contaminants are IgG (**) and the major coat protein of the yeast virus L-A (*). The identity of each band was confirmed by mass spectrometry. ( B ) The VFT complex, prepared from cells expressing Vps53-PtA and Vps54p-myc (strain SSY16) and purified by IgG–Sepharose, was incubated with GST–Ypt6p in the GTPγS or GDP form, eluted and detected with anti-myc antibodies as described in Materials and methods. The VFT lane is equivalent to 15% of the input material.
Figure Legend Snippet: Fig. 3. The VFT complex binds directly to Ypt6p. ( A ) Purification of the VFT complex from yeast cells expressing Vps54p-PtA (strain SSY14). The complex was eluted from IgG–Sepharose with TEV protease, which cleaved off the PtA. Besides the protease (TEV), the contaminants are IgG (**) and the major coat protein of the yeast virus L-A (*). The identity of each band was confirmed by mass spectrometry. ( B ) The VFT complex, prepared from cells expressing Vps53-PtA and Vps54p-myc (strain SSY16) and purified by IgG–Sepharose, was incubated with GST–Ypt6p in the GTPγS or GDP form, eluted and detected with anti-myc antibodies as described in Materials and methods. The VFT lane is equivalent to 15% of the input material.

Techniques Used: Purification, Expressing, Mass Spectrometry, Incubation

Fig. 6. The VFT complex can be cross-linked to late Golgi SNAREs in vivo . ( A ) Spheroplasts of cells expressing either Vps54-PtA (strain SSY14) or PtA (strain SSY17) were treated with 0.5, 1 or 3 mM of the cross-linker (DSP), detergent solubilized and the PtA fusion proteins affinity purified on IgG–Sepharose. Samples from the detergent-solubilized extracts (E, shown here for the 0.5 mM DSP extract) or the purified fractions (IP) were analysed by western blotting using anti-Tlg1p, Tlg2p, Sed5p, Sso2p and PtA antibodies. Note that the IP samples are overloaded relative to the E samples, as shown by the PtA blot. ( B ) Cells with (+) or without (–) YPT6 , expressing Vps54p-PtA (strains SSY14 or SSY18, respectively) or PtA (SSY17), were spheroplasted, cross-linked with 1 mM DSP and analysed as in (A). ( C ) Cells expressing Vps54p-myc, PtA-Tlg1p (from centromere plasmids) and YPT6 as indicated (from left to right, strains SSY21, SSY20 and SSY19) were spheroplasted, cross-linked with 1 mM DSP, and PtA-Tlg1p was affinity purified on IgG–Sepharose as in (A). Samples from the extracts (E) and the affinity-purified fractions (IP) were analysed by western blotting using anti-myc and anti-Imh1p antibodies.
Figure Legend Snippet: Fig. 6. The VFT complex can be cross-linked to late Golgi SNAREs in vivo . ( A ) Spheroplasts of cells expressing either Vps54-PtA (strain SSY14) or PtA (strain SSY17) were treated with 0.5, 1 or 3 mM of the cross-linker (DSP), detergent solubilized and the PtA fusion proteins affinity purified on IgG–Sepharose. Samples from the detergent-solubilized extracts (E, shown here for the 0.5 mM DSP extract) or the purified fractions (IP) were analysed by western blotting using anti-Tlg1p, Tlg2p, Sed5p, Sso2p and PtA antibodies. Note that the IP samples are overloaded relative to the E samples, as shown by the PtA blot. ( B ) Cells with (+) or without (–) YPT6 , expressing Vps54p-PtA (strains SSY14 or SSY18, respectively) or PtA (SSY17), were spheroplasted, cross-linked with 1 mM DSP and analysed as in (A). ( C ) Cells expressing Vps54p-myc, PtA-Tlg1p (from centromere plasmids) and YPT6 as indicated (from left to right, strains SSY21, SSY20 and SSY19) were spheroplasted, cross-linked with 1 mM DSP, and PtA-Tlg1p was affinity purified on IgG–Sepharose as in (A). Samples from the extracts (E) and the affinity-purified fractions (IP) were analysed by western blotting using anti-myc and anti-Imh1p antibodies.

Techniques Used: In Vivo, Expressing, Affinity Purification, Purification, Western Blot

Fig. 4. Binding of the VFT complex to SNAREs. ( A ) Binding of Vps54p-PtA from cytosol to glutathione–Sepharose beads containing the indicated GST–SNARE fusions. The cytosol lane is equivalent to 5% of the material added to the binding reactions. The last three lanes are from a separate experiment; Tlg1-N contains amino acid residues 1–131 of Tlg1p; Tlg1-C contains residues 132–206. ( B ) As (A), but purified VFT complex, prepared as in Figure 3A, was used for the binding.
Figure Legend Snippet: Fig. 4. Binding of the VFT complex to SNAREs. ( A ) Binding of Vps54p-PtA from cytosol to glutathione–Sepharose beads containing the indicated GST–SNARE fusions. The cytosol lane is equivalent to 5% of the material added to the binding reactions. The last three lanes are from a separate experiment; Tlg1-N contains amino acid residues 1–131 of Tlg1p; Tlg1-C contains residues 132–206. ( B ) As (A), but purified VFT complex, prepared as in Figure 3A, was used for the binding.

Techniques Used: Binding Assay, Purification

53) Product Images from "Notch3 Interactome Analysis Identified WWP2 as a Negative Regulator of Notch3 Signaling in Ovarian Cancer"

Article Title: Notch3 Interactome Analysis Identified WWP2 as a Negative Regulator of Notch3 Signaling in Ovarian Cancer

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1004751

WWP2 ubiquitinates Notch3 receptor and regulates its activity. ( A ) Schematic representation of N3-TM, N3-NEXT, and N3-ICD constructs. Signal peptide sequences were inserted into N3-TM and N3-NEXT constructs. To facilitate detection, all Notch3 variant constructs contained a V5 epitope tag at the C-terminus. ( B ) 293T cells were transfected with HA-tagged ubiquitin together WWP2 and different Notch3 variant constructs: N3-TM, N3-NEXT, or N3-ICD. To determine the expression of different constructs, Western blot analysis was performed with anti-V5, anti-FLAG, or anti-HA, antibody using input lysates. Equal loading was determined with an anti-GAPDH antibody. ( C ) To detect ubiquitinated Notch3, immunoprecipitation (IP) was performed on the cell lysates using anti-V5 beads, and western blot (WB) was performed with an anti-HA antibody. To detect the interaction between Notch3 variant and WWP2, IP was performed by using anti-Flag agarose, and western blot was performed using anti-V5 antibody. Left panel: short exposure; right panel: longer exposure. ( D ) Schematic representation of wild type WWP2, a catalytically inactive C838A mutant of WWP2 (WWP2-CA), and a deletion construct of WWP2 lacking the HECT ubiquitination domain and three WW domains (WWP2ΔHECT). All of the WWP2 expression constructs contained a flag epitope tag at the C-terminal end. ( E ) 293 cells were transfected with N3-NEXT, ubiquitin-HA, and pLPC control plasmid, WWP2, WWP2-CA, or WWP2ΔHECT. To determine expression of the different constructs, western blot analysis of lysates was performed with anti-FLAG or anti-V5 antibody (input lysate). ( F ) To detect ubiquitinated N3-NEXT, immunoprecipitation (IP) was performed on the cell lysates using the anti-V5 beads, and western blot (WB) was performed with an anti-HA antibody. Reciprocal co-IP was performed by using anti-HA agarose beads for pull-down and using the anti-V5 antibody for western blot.
Figure Legend Snippet: WWP2 ubiquitinates Notch3 receptor and regulates its activity. ( A ) Schematic representation of N3-TM, N3-NEXT, and N3-ICD constructs. Signal peptide sequences were inserted into N3-TM and N3-NEXT constructs. To facilitate detection, all Notch3 variant constructs contained a V5 epitope tag at the C-terminus. ( B ) 293T cells were transfected with HA-tagged ubiquitin together WWP2 and different Notch3 variant constructs: N3-TM, N3-NEXT, or N3-ICD. To determine the expression of different constructs, Western blot analysis was performed with anti-V5, anti-FLAG, or anti-HA, antibody using input lysates. Equal loading was determined with an anti-GAPDH antibody. ( C ) To detect ubiquitinated Notch3, immunoprecipitation (IP) was performed on the cell lysates using anti-V5 beads, and western blot (WB) was performed with an anti-HA antibody. To detect the interaction between Notch3 variant and WWP2, IP was performed by using anti-Flag agarose, and western blot was performed using anti-V5 antibody. Left panel: short exposure; right panel: longer exposure. ( D ) Schematic representation of wild type WWP2, a catalytically inactive C838A mutant of WWP2 (WWP2-CA), and a deletion construct of WWP2 lacking the HECT ubiquitination domain and three WW domains (WWP2ΔHECT). All of the WWP2 expression constructs contained a flag epitope tag at the C-terminal end. ( E ) 293 cells were transfected with N3-NEXT, ubiquitin-HA, and pLPC control plasmid, WWP2, WWP2-CA, or WWP2ΔHECT. To determine expression of the different constructs, western blot analysis of lysates was performed with anti-FLAG or anti-V5 antibody (input lysate). ( F ) To detect ubiquitinated N3-NEXT, immunoprecipitation (IP) was performed on the cell lysates using the anti-V5 beads, and western blot (WB) was performed with an anti-HA antibody. Reciprocal co-IP was performed by using anti-HA agarose beads for pull-down and using the anti-V5 antibody for western blot.

Techniques Used: Activity Assay, Construct, Variant Assay, Transfection, Expressing, Western Blot, Immunoprecipitation, Mutagenesis, FLAG-tag, Plasmid Preparation, Co-Immunoprecipitation Assay

WWP2 interacts with endogenous Notch3 in cancer cells. ( A ) MCF7 cells were incubated with 25 mM NH4Cl for 0, 30, 60, 120, and 240 minutes. Cells were lysed and the lysates separated into nuclear (N) and cytosol/membrane (C) fractions. Western blot analysis was performed with an anti-Notch3 antibody. Equal loading was determined with an anti-GAPDH antibody for the cytosol/membrane fraction and with an anti-histone 3 antibody for the nuclear fraction. The star represents the N3-NEXT bands. ( B ) MCF7 cells were incubated with 2.5 mM EDTA for different times as indicated to induce Notch-ICD generation. Cells were lysed and fractionated into nuclear (N) and cytosol/membrane (C) fractions. Western blot analysis was performed with an anti-Notch3 antibody. ( C ) Ovarian cancer cells were first transfected with the flag tagged WWP2 expressing plasmids. The cells were then treated with 2.5 mM EDTA for 20 minutes or with 25 mM NH4Cl for 240 minutes, and then fractionated into nuclear and cytosol/membrane fractions. Immunoprecipitation (IP) was performed with anti-flag agarose beads for pull-down and a rabbit anti-Notch3 antibody for western blot. Expression of GAPDH and histones were used for demonstrating the purity of nuclear and cytosol/membrane fractions, respectively.
Figure Legend Snippet: WWP2 interacts with endogenous Notch3 in cancer cells. ( A ) MCF7 cells were incubated with 25 mM NH4Cl for 0, 30, 60, 120, and 240 minutes. Cells were lysed and the lysates separated into nuclear (N) and cytosol/membrane (C) fractions. Western blot analysis was performed with an anti-Notch3 antibody. Equal loading was determined with an anti-GAPDH antibody for the cytosol/membrane fraction and with an anti-histone 3 antibody for the nuclear fraction. The star represents the N3-NEXT bands. ( B ) MCF7 cells were incubated with 2.5 mM EDTA for different times as indicated to induce Notch-ICD generation. Cells were lysed and fractionated into nuclear (N) and cytosol/membrane (C) fractions. Western blot analysis was performed with an anti-Notch3 antibody. ( C ) Ovarian cancer cells were first transfected with the flag tagged WWP2 expressing plasmids. The cells were then treated with 2.5 mM EDTA for 20 minutes or with 25 mM NH4Cl for 240 minutes, and then fractionated into nuclear and cytosol/membrane fractions. Immunoprecipitation (IP) was performed with anti-flag agarose beads for pull-down and a rabbit anti-Notch3 antibody for western blot. Expression of GAPDH and histones were used for demonstrating the purity of nuclear and cytosol/membrane fractions, respectively.

Techniques Used: Incubation, Western Blot, Transfection, Expressing, Immunoprecipitation

54) Product Images from "The Toxoplasma gondii dense granule protein TgGRA3 interacts with host Golgi and dysregulates anterograde transport"

Article Title: The Toxoplasma gondii dense granule protein TgGRA3 interacts with host Golgi and dysregulates anterograde transport

Journal: Biology Open

doi: 10.1242/bio.039818

TgGRA3 interacts with TgGRA23 but not TgROP13. (A) Representative z-stack images from confocal microscopy showing nuclei (DAPI, blue), TgGRA3 (green), and TgGRA23_3×HA (red). Scale bars: 10 µm. (B) Percentage of colocalization between TgGRA3 and TgGRA23_3×HA or TgROP13_3×HA was measured after three-dimensional reconstruction of z-stack images ( n =16 parasitophorous vacuoles). Results are reported as means±s.d. (C) Pearson's correlation coefficient from data in B confirmed partial co-localization between TgGRA3 and TgGRA23. Results are reported in±s.d. (D) Western blot representative of three independent experiments showing immunoprecipitation of TgGRA3 by TgGRA23_3×HA. TgGRA23_3×HA and TgROP13_3×HA lysates were incubated with anti-HA antibodies coupled to Sepharose beads, followed by immunoblotting for TgGRA3, TgGRA23_3×HA, and TgROP13_3×HA. (E) Quantification of immunoprecipitation carried out in D. Results from three independent experiments are represented. Results are reported as means±s.d. * P =0.0311 (Welch's t -test). (F) Immunoprecipitation of TgGRA23_3×HA in parasite extracts (low-speed supernatant: LSP) and PVM extracts (high-speed supernatant: HSP). TgGRA3 and TgGRA23_3×HA were detected by immunoblotting. The parasite nuclear marker TgENO2 was used as a negative control. (G) Binding of TgGRA23_3×HA to GST-GRA3 43-161 and GST-Δ110-128 versus GST alone. Bound TgGRA23_3×HA was detected by immunoblotting with anti-HA antibodies. Equal quantities of TgGRA3 proteins were confirmed by Coomassie Blue staining. This blot shows no binding of TgGRA23_3×HA to GST-Δ110-128. (H) Histogram quantifying different host Golgi localizations in cells infected by Δ GRA23 or Δ ROP13 parasites versus RH Δ KU80 and Δ GRA3 parasites. Golgi localizations were classified as not recruited (far), localized above the PV (top) or surrounding the PV (around). For each condition, 300 parasitophorous vacuoles from three independent experiments were counted. * P
Figure Legend Snippet: TgGRA3 interacts with TgGRA23 but not TgROP13. (A) Representative z-stack images from confocal microscopy showing nuclei (DAPI, blue), TgGRA3 (green), and TgGRA23_3×HA (red). Scale bars: 10 µm. (B) Percentage of colocalization between TgGRA3 and TgGRA23_3×HA or TgROP13_3×HA was measured after three-dimensional reconstruction of z-stack images ( n =16 parasitophorous vacuoles). Results are reported as means±s.d. (C) Pearson's correlation coefficient from data in B confirmed partial co-localization between TgGRA3 and TgGRA23. Results are reported in±s.d. (D) Western blot representative of three independent experiments showing immunoprecipitation of TgGRA3 by TgGRA23_3×HA. TgGRA23_3×HA and TgROP13_3×HA lysates were incubated with anti-HA antibodies coupled to Sepharose beads, followed by immunoblotting for TgGRA3, TgGRA23_3×HA, and TgROP13_3×HA. (E) Quantification of immunoprecipitation carried out in D. Results from three independent experiments are represented. Results are reported as means±s.d. * P =0.0311 (Welch's t -test). (F) Immunoprecipitation of TgGRA23_3×HA in parasite extracts (low-speed supernatant: LSP) and PVM extracts (high-speed supernatant: HSP). TgGRA3 and TgGRA23_3×HA were detected by immunoblotting. The parasite nuclear marker TgENO2 was used as a negative control. (G) Binding of TgGRA23_3×HA to GST-GRA3 43-161 and GST-Δ110-128 versus GST alone. Bound TgGRA23_3×HA was detected by immunoblotting with anti-HA antibodies. Equal quantities of TgGRA3 proteins were confirmed by Coomassie Blue staining. This blot shows no binding of TgGRA23_3×HA to GST-Δ110-128. (H) Histogram quantifying different host Golgi localizations in cells infected by Δ GRA23 or Δ ROP13 parasites versus RH Δ KU80 and Δ GRA3 parasites. Golgi localizations were classified as not recruited (far), localized above the PV (top) or surrounding the PV (around). For each condition, 300 parasitophorous vacuoles from three independent experiments were counted. * P

Techniques Used: Confocal Microscopy, Western Blot, Immunoprecipitation, Incubation, Marker, Negative Control, Binding Assay, Staining, Infection

Inducible TgGRA3 depletion affects host Golgi recruitment and entry at the PVM. (A) Agarose gel of PCR showing correct integration of the transgene into Tg GRA3 locus. SOD (superoxide dismutase) is used as a PCR loading control. (B) Western blot analysis demonstrating the decrease in TgGRA3 protein level in iKD-GRA3 mutants. ATc treatment depleted TgGRA3 protein expression in iKD-GRA3 mutants. Eno2 is used as a loading control. (C) Representative z-stack from confocal microscopy images of HFF cells infected with RH TaTi parental strain and iKD-GRA3 in absence or in presence of ATc. At 35 h post-infection, iKD-GRA3 parasites indicated abnormal accumulations of host Golgi material at the PVM (white dashed squares). A magnification of this region is presented in the lower panel. The host Golgi marker giantin (red), PV marker TgGRA5 (green), and nuclei (DAPI) were stained. Scale bars: 10 µm (5 µm in magnified regions). (D) Histogram quantifying PV invagination volume. Invagination volumes ( n =80) were measured from three-dimensional reconstructions of 30 parasitophorous vacuoles for each condition, ** P value
Figure Legend Snippet: Inducible TgGRA3 depletion affects host Golgi recruitment and entry at the PVM. (A) Agarose gel of PCR showing correct integration of the transgene into Tg GRA3 locus. SOD (superoxide dismutase) is used as a PCR loading control. (B) Western blot analysis demonstrating the decrease in TgGRA3 protein level in iKD-GRA3 mutants. ATc treatment depleted TgGRA3 protein expression in iKD-GRA3 mutants. Eno2 is used as a loading control. (C) Representative z-stack from confocal microscopy images of HFF cells infected with RH TaTi parental strain and iKD-GRA3 in absence or in presence of ATc. At 35 h post-infection, iKD-GRA3 parasites indicated abnormal accumulations of host Golgi material at the PVM (white dashed squares). A magnification of this region is presented in the lower panel. The host Golgi marker giantin (red), PV marker TgGRA5 (green), and nuclei (DAPI) were stained. Scale bars: 10 µm (5 µm in magnified regions). (D) Histogram quantifying PV invagination volume. Invagination volumes ( n =80) were measured from three-dimensional reconstructions of 30 parasitophorous vacuoles for each condition, ** P value

Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Western Blot, Expressing, Confocal Microscopy, Infection, Marker, Staining

55) Product Images from "The Gcn2 Regulator Yih1 Interacts with the Cyclin Dependent Kinase Cdc28 and Promotes Cell Cycle Progression through G2/M in Budding Yeast"

Article Title: The Gcn2 Regulator Yih1 Interacts with the Cyclin Dependent Kinase Cdc28 and Promotes Cell Cycle Progression through G2/M in Budding Yeast

Journal: PLoS ONE

doi: 10.1371/journal.pone.0131070

Cdc28 co-precipitates with GST-Yih1. (A) In vivo GST-pull-down assay. yih1Δ strains (MSY-Y2) expressing GST-Yih1 or GST alone from the galactose inducible promoter were grown to log-phase and harvested. Equal amounts of WCEs (2 mg) were subjected to glutathione-mediated GST pull-down assays. The precipitates (100% of the bound proteins – right-panel) and the input (1/100 th of the input – left panel) were assessed by immunoblot to detect the indicated proteins. (B) GST-Yih1 purified from E . coli co-precipitates endogenous Cdc28 from yeast WCEs. Full-length Yih1 fused to GST or GST alone were expressed in E . coli , purified, immobilized on glutathione-Sepharose beads and incubated with equal amounts of glutathione-Sepharose pre-cleared WCEs (1.25 mg) prepared from a yih1Δ strain (MSY-Y2). After extensive washes the precipitates (100% of the bound proteins) and the input (1/50 th of the input) were analyzed by immunoblot to detect the indicated proteins. The Ponceau staining of the membrane is shown (lower panel). One representative blot from two independent experiments performed in duplicate is shown.
Figure Legend Snippet: Cdc28 co-precipitates with GST-Yih1. (A) In vivo GST-pull-down assay. yih1Δ strains (MSY-Y2) expressing GST-Yih1 or GST alone from the galactose inducible promoter were grown to log-phase and harvested. Equal amounts of WCEs (2 mg) were subjected to glutathione-mediated GST pull-down assays. The precipitates (100% of the bound proteins – right-panel) and the input (1/100 th of the input – left panel) were assessed by immunoblot to detect the indicated proteins. (B) GST-Yih1 purified from E . coli co-precipitates endogenous Cdc28 from yeast WCEs. Full-length Yih1 fused to GST or GST alone were expressed in E . coli , purified, immobilized on glutathione-Sepharose beads and incubated with equal amounts of glutathione-Sepharose pre-cleared WCEs (1.25 mg) prepared from a yih1Δ strain (MSY-Y2). After extensive washes the precipitates (100% of the bound proteins) and the input (1/50 th of the input) were analyzed by immunoblot to detect the indicated proteins. The Ponceau staining of the membrane is shown (lower panel). One representative blot from two independent experiments performed in duplicate is shown.

Techniques Used: In Vivo, Pull Down Assay, Expressing, Purification, Incubation, Staining

Mammalian IMPACT forms a complex with Cdc28 and CDK1. (A) Mammalian IMPACT expressed in yeast precipitates Cdc28. Two different transformants of yih1Δ strain (BY4741) expressing either GST-IMPACT or GST alone from a galactose inducible promoter were grown to log phase in SGal. WCEs were prepared and equivalent amounts of protein (1 mg) were subjected to GST-pull-down assays. The precipitated complexes were analyzed by immunoblot for the indicated proteins. The input lanes (left-panel) contained 4% of the WCEs used in the assay. (B) CDK1 co-precipitates with Flag-tagged IMPACT in N2a cells. Undifferentiated mouse N2a cells were transfected with a plasmid expressing IMPACT fused to Flag or with the vector alone (pFLAG). Cell lysates were cleared with protein-A agarose and subjected to immunoprecipitation with anti-Flag antibodies (M2-Flag-Resin). All the precipitated material and 1% of the input material were subjected to immunoblot to detect Flag-IMPACT, CDK1, and GAPDH as negative control.
Figure Legend Snippet: Mammalian IMPACT forms a complex with Cdc28 and CDK1. (A) Mammalian IMPACT expressed in yeast precipitates Cdc28. Two different transformants of yih1Δ strain (BY4741) expressing either GST-IMPACT or GST alone from a galactose inducible promoter were grown to log phase in SGal. WCEs were prepared and equivalent amounts of protein (1 mg) were subjected to GST-pull-down assays. The precipitated complexes were analyzed by immunoblot for the indicated proteins. The input lanes (left-panel) contained 4% of the WCEs used in the assay. (B) CDK1 co-precipitates with Flag-tagged IMPACT in N2a cells. Undifferentiated mouse N2a cells were transfected with a plasmid expressing IMPACT fused to Flag or with the vector alone (pFLAG). Cell lysates were cleared with protein-A agarose and subjected to immunoprecipitation with anti-Flag antibodies (M2-Flag-Resin). All the precipitated material and 1% of the input material were subjected to immunoblot to detect Flag-IMPACT, CDK1, and GAPDH as negative control.

Techniques Used: Expressing, Transfection, Plasmid Preparation, Immunoprecipitation, Negative Control

56) Product Images from "DNA-PKcs phosphorylates hnRNP-A1 to facilitate the RPA-to-POT1 switch and telomere capping after replication"

Article Title: DNA-PKcs phosphorylates hnRNP-A1 to facilitate the RPA-to-POT1 switch and telomere capping after replication

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkv539

Phosphomimetics facilitate hnRNP-A1 mutants binding to single-stranded telomeric DNA. ( A ) The association between GST-H3 DNA-PKcs fragment and hnRNP-A1 protein was challenged with increasing concentrations (0.2 μM, 2 μM) of either signal-strand telomeric DNA (ssTEL, TTAGGG x8 ) or mutant DNA (ssMUT, TTTGCG x8 ). The bound hnRNP-A1 proteins were western blotted with anti-His antibody. ( B ) WT and mutant hnRNP-A1 proteins were incubated with both GST-H3 fusion protein and biotinylated ssTEL (0.5 μM). GST-H3 bound hnRNP-A1 was retrieved by Glutathion-sepharose beads. The unbound hnRNPA1:ssTEL complex was subsequently retrieved by streptavidin beads and analyzed by western blot. ( C ) Recombinant hnRNP-A1 proteins were incubated with either ssTEL or ssMUT oligonucleotides and analyzed in electrophoretic mobility shift assay (EMSA).
Figure Legend Snippet: Phosphomimetics facilitate hnRNP-A1 mutants binding to single-stranded telomeric DNA. ( A ) The association between GST-H3 DNA-PKcs fragment and hnRNP-A1 protein was challenged with increasing concentrations (0.2 μM, 2 μM) of either signal-strand telomeric DNA (ssTEL, TTAGGG x8 ) or mutant DNA (ssMUT, TTTGCG x8 ). The bound hnRNP-A1 proteins were western blotted with anti-His antibody. ( B ) WT and mutant hnRNP-A1 proteins were incubated with both GST-H3 fusion protein and biotinylated ssTEL (0.5 μM). GST-H3 bound hnRNP-A1 was retrieved by Glutathion-sepharose beads. The unbound hnRNPA1:ssTEL complex was subsequently retrieved by streptavidin beads and analyzed by western blot. ( C ) Recombinant hnRNP-A1 proteins were incubated with either ssTEL or ssMUT oligonucleotides and analyzed in electrophoretic mobility shift assay (EMSA).

Techniques Used: Binding Assay, Mutagenesis, Western Blot, Incubation, Recombinant, Electrophoretic Mobility Shift Assay

Association between DNA-PKcs and hnRNP-A1 in vivo and in vitro . ( A ) Asynchronous and nocodazole synchronized HeLa cell lysates were IP with control IgG or specific antibodies against DNA-PKcs (PKcs) or hnRNP-A1 (A1) followed by western blotting. H3 pS10 was used as markers for G2/M synchronization (right panel). ( B ) Similar co-IP analysis was performed in HCT116 cells. ( C ) Schematic of GST fusions with various DNA-PKcs fragments. The C-terminal DNA-PKcs fragment ‘H’ including the kinase domain (KD), PIKK-regulatory domain (P) and FAT-C-terminal domain (F) was further divided into H1-H3 fragments. ( D ) GST-DNA-PKcs fragments were incubated with His-tagged full-length hnRNP-A1 followed by retrieval with Glutathion-sepharose beads. The bound hnRNP-A1 was western blotted with anti-His antibody (bottom panel). The loading of various GST fusions was demonstrated by Ponceau S staining (top panel) and anti-GST antibody (middle panel). ( E ) DNA-PKcs C-terminal fragments directly interact with full-length hnRNPA1. Asterisks indicate GST-DNA-PKcs fusions by Ponceau S staining (top panel). ( F ) Schematic of hnRNP-A1 contains two RNA binding motifs (RRMs), Gly-rich (Gly) and nucleocytoplasmic shuttling (M9) domains. GST-H3 fusion of DNA-PKcs preferentially interacts with the RRMs (R) but not the Gly-M9 (GM) fragment of hnRNPA1.
Figure Legend Snippet: Association between DNA-PKcs and hnRNP-A1 in vivo and in vitro . ( A ) Asynchronous and nocodazole synchronized HeLa cell lysates were IP with control IgG or specific antibodies against DNA-PKcs (PKcs) or hnRNP-A1 (A1) followed by western blotting. H3 pS10 was used as markers for G2/M synchronization (right panel). ( B ) Similar co-IP analysis was performed in HCT116 cells. ( C ) Schematic of GST fusions with various DNA-PKcs fragments. The C-terminal DNA-PKcs fragment ‘H’ including the kinase domain (KD), PIKK-regulatory domain (P) and FAT-C-terminal domain (F) was further divided into H1-H3 fragments. ( D ) GST-DNA-PKcs fragments were incubated with His-tagged full-length hnRNP-A1 followed by retrieval with Glutathion-sepharose beads. The bound hnRNP-A1 was western blotted with anti-His antibody (bottom panel). The loading of various GST fusions was demonstrated by Ponceau S staining (top panel) and anti-GST antibody (middle panel). ( E ) DNA-PKcs C-terminal fragments directly interact with full-length hnRNPA1. Asterisks indicate GST-DNA-PKcs fusions by Ponceau S staining (top panel). ( F ) Schematic of hnRNP-A1 contains two RNA binding motifs (RRMs), Gly-rich (Gly) and nucleocytoplasmic shuttling (M9) domains. GST-H3 fusion of DNA-PKcs preferentially interacts with the RRMs (R) but not the Gly-M9 (GM) fragment of hnRNPA1.

Techniques Used: In Vivo, In Vitro, Western Blot, Co-Immunoprecipitation Assay, Incubation, Staining, RNA Binding Assay

57) Product Images from "Specific Cooperation Between Imp-?2 and Imp-?/Ketel in Spindle Assembly During Drosophila Early Nuclear Divisions"

Article Title: Specific Cooperation Between Imp-?2 and Imp-?/Ketel in Spindle Assembly During Drosophila Early Nuclear Divisions

Journal: G3: Genes|Genomes|Genetics

doi: 10.1534/g3.111.001073

Imp-β KetD and Imp-β KetRE34 bind RanGDP and RanGTP with a higher affinity than wild-type Imp-β. His-RanT 24 N (left panel) and His-RanQ 69 L (right panel) proteins, representing the GDP- and GTP-bound forms, respectively, were expressed in bacteria, purified, and subsequently added to wild-type embryonic protein extract. Aliquots of both mixtures were incubated with GST-Imp-β, and either GST-Imp-β KetD or GST-Imp-β KetRE34 fusion proteins immobilized on Glutathione Sepharose beads. Proteins bound to the beads were analyzed by SDS-PAGE and immune-detected on Western blot with anti-Ran and anti-Imp-β antibodies.
Figure Legend Snippet: Imp-β KetD and Imp-β KetRE34 bind RanGDP and RanGTP with a higher affinity than wild-type Imp-β. His-RanT 24 N (left panel) and His-RanQ 69 L (right panel) proteins, representing the GDP- and GTP-bound forms, respectively, were expressed in bacteria, purified, and subsequently added to wild-type embryonic protein extract. Aliquots of both mixtures were incubated with GST-Imp-β, and either GST-Imp-β KetD or GST-Imp-β KetRE34 fusion proteins immobilized on Glutathione Sepharose beads. Proteins bound to the beads were analyzed by SDS-PAGE and immune-detected on Western blot with anti-Ran and anti-Imp-β antibodies.

Techniques Used: Purification, Incubation, SDS Page, Western Blot

Isolation of Drosophila ovarian proteins specifically associated with the NLSB domain of Imp-α2. Proteins were extracted from ovaries of transformed flies producing zz-tagged Imp-α2 (Imp-α2zz) or zz-tagged NLSB − Imp-α2 (NLSB − zz). (A) SDS-polyacrylamide gel stained with Coomassie Blue for proteins from high-speed supernatants (HSS) of control and Imp-α2zz extracts (left two lanes). The proteins show equal distribution in both extracts. The HSS proteins were then adsorbed on IgG Sepharose beads, and the eluted proteins were separated on SDS-polyacrylamide gel (right two lanes). (B) The procedure was repeated for Imp-α2zz and NLSB − zz ovarian extracts. Protein bands present in the Imp-α2zz purified fraction but absent from the NLSB − zz fraction were excised, digested with trypsin and subjected to mass spectrometry. The following proteins were identified in the selected bands: (1) CP190, (2) ISWI, and (3) lamin Dm0.
Figure Legend Snippet: Isolation of Drosophila ovarian proteins specifically associated with the NLSB domain of Imp-α2. Proteins were extracted from ovaries of transformed flies producing zz-tagged Imp-α2 (Imp-α2zz) or zz-tagged NLSB − Imp-α2 (NLSB − zz). (A) SDS-polyacrylamide gel stained with Coomassie Blue for proteins from high-speed supernatants (HSS) of control and Imp-α2zz extracts (left two lanes). The proteins show equal distribution in both extracts. The HSS proteins were then adsorbed on IgG Sepharose beads, and the eluted proteins were separated on SDS-polyacrylamide gel (right two lanes). (B) The procedure was repeated for Imp-α2zz and NLSB − zz ovarian extracts. Protein bands present in the Imp-α2zz purified fraction but absent from the NLSB − zz fraction were excised, digested with trypsin and subjected to mass spectrometry. The following proteins were identified in the selected bands: (1) CP190, (2) ISWI, and (3) lamin Dm0.

Techniques Used: Isolation, Transformation Assay, Staining, Purification, Mass Spectrometry

58) Product Images from "HIV-1 Rev protein specifies the viral RNA export pathway by suppressing TAP/NXF1 recruitment"

Article Title: HIV-1 Rev protein specifies the viral RNA export pathway by suppressing TAP/NXF1 recruitment

Journal: Nucleic Acids Research

doi: 10.1093/nar/gku304

Effect of Rev on the association of mRNA binding proteins. ( A ) The same 32 P-labeled RNA mixture as in Figure 1 was injected into the nucleus in the absence or presence of Rev. The nuclear fraction was prepared after 1 h, and GST pull-down was performed with glutathione beads that had been pre-bound with either the GST-TAP231 or GST protein. RNA precipitated with each type of bead was recovered and analyzed. The input lanes were loaded with 10% of each input mixture. ( B ) The nuclear fraction was prepared as in (A), and IP was performed with the anti-Aly/REF monoclonal antibody (11G5, αAly), anti-Y14 monoclonal antibody (4C4, αY14) or anti-Myc monoclonal antibody (9E10, αMyc) that had been pre-bound to Protein A-Sepharose beads. RNA precipitated with each antibody was recovered and analyzed. ( C ) The recombinant FLAG-UAP56 protein (50 fmol/oocyte) was pre-injected into the cytoplasm. After 16 h incubation, a second microinjection was performed into the nucleus with the same 32 P-labeled RNA mixture as in Figure 1 , except that pre-ftz RNA was used instead of elongated U1 RNA, in the absence or presence of Rev. IP was performed with 11G5, the anti-FLAG monoclonal antibody (M2, αFLAG), or 9E10. ( D ) Rev inhibits the association of TAP and the TREX complex with spliced ftzRRE RNA.
Figure Legend Snippet: Effect of Rev on the association of mRNA binding proteins. ( A ) The same 32 P-labeled RNA mixture as in Figure 1 was injected into the nucleus in the absence or presence of Rev. The nuclear fraction was prepared after 1 h, and GST pull-down was performed with glutathione beads that had been pre-bound with either the GST-TAP231 or GST protein. RNA precipitated with each type of bead was recovered and analyzed. The input lanes were loaded with 10% of each input mixture. ( B ) The nuclear fraction was prepared as in (A), and IP was performed with the anti-Aly/REF monoclonal antibody (11G5, αAly), anti-Y14 monoclonal antibody (4C4, αY14) or anti-Myc monoclonal antibody (9E10, αMyc) that had been pre-bound to Protein A-Sepharose beads. RNA precipitated with each antibody was recovered and analyzed. ( C ) The recombinant FLAG-UAP56 protein (50 fmol/oocyte) was pre-injected into the cytoplasm. After 16 h incubation, a second microinjection was performed into the nucleus with the same 32 P-labeled RNA mixture as in Figure 1 , except that pre-ftz RNA was used instead of elongated U1 RNA, in the absence or presence of Rev. IP was performed with 11G5, the anti-FLAG monoclonal antibody (M2, αFLAG), or 9E10. ( D ) Rev inhibits the association of TAP and the TREX complex with spliced ftzRRE RNA.

Techniques Used: Binding Assay, Labeling, Injection, Recombinant, Incubation

Effect of TAP-p15 overexpression on HIV-1 expression. ( A ) Genome organization of HIV-1 NL4-3ΔRev. The start codon of the Rev gene was mutated from ATG to ACG. ( B ) HEK293T cells in a 6-well plate (70% confluent) were transfected with pcDNA3-GFP (1 μg), pNL4-3ΔRev (1 μg), and either pCI-neo (0.1 μg) (−) or pCI-FLAG-Rev (0.1 μg) (+). After 24 h, cells were collected and proteins from cell pellets were analyzed by SDS-PAGE and western blotting with rabbit anti-Gag p55 antiserum. UT: untransfected cells were used as a control. ( C ) HEK293T cells in a 6-well plate (70% confluent) were transfected with pcDNA3-GFP (1 μg), pNL4-3ΔRev (1 μg), pCI-FLAG-Rev (0.1 or 1 μg), and either pcDNA5 (1 μg) (−) or pcDNA5-FLAG-TAP (0.6 μg) plus pcDNA5-FLAG-p15 (0.3 μg) (+). 0.9 μg of pCI-neo was added for 0.1 μg of pCI-FLAG-Rev to equalize the amount of plasmid DNAs. Supernatants and cells were collected after 24 h. RNA from cell pellets was subjected to semi-quantitative RT-PCR. PCR products were analyzed by electrophoresis in a 2% agarose gel. ( D ) Quantitation of the relative level of RNAs from three independent experiments performed as in (C). GFP mRNA was used for normalization. 0.1 μg of the pCI-FLAG-Rev sample (lane 1) was set to 1. Averages and standard deviations are shown. ( E ) Protein from the cell pellets in (C) was analyzed by SDS-PAGE and western blotting with rabbit anti-Gag p55 antiserum or the anti-GFP antibody. UT: untransfected cells were used as a control. U2AF 65 was a loading control. ( F ) The filtrated media from (C) were immunoprecipitated with rabbit anti-Gag p55 antiserum and detected by western blotting with the monoclonal anti-Gag p24 antibody.
Figure Legend Snippet: Effect of TAP-p15 overexpression on HIV-1 expression. ( A ) Genome organization of HIV-1 NL4-3ΔRev. The start codon of the Rev gene was mutated from ATG to ACG. ( B ) HEK293T cells in a 6-well plate (70% confluent) were transfected with pcDNA3-GFP (1 μg), pNL4-3ΔRev (1 μg), and either pCI-neo (0.1 μg) (−) or pCI-FLAG-Rev (0.1 μg) (+). After 24 h, cells were collected and proteins from cell pellets were analyzed by SDS-PAGE and western blotting with rabbit anti-Gag p55 antiserum. UT: untransfected cells were used as a control. ( C ) HEK293T cells in a 6-well plate (70% confluent) were transfected with pcDNA3-GFP (1 μg), pNL4-3ΔRev (1 μg), pCI-FLAG-Rev (0.1 or 1 μg), and either pcDNA5 (1 μg) (−) or pcDNA5-FLAG-TAP (0.6 μg) plus pcDNA5-FLAG-p15 (0.3 μg) (+). 0.9 μg of pCI-neo was added for 0.1 μg of pCI-FLAG-Rev to equalize the amount of plasmid DNAs. Supernatants and cells were collected after 24 h. RNA from cell pellets was subjected to semi-quantitative RT-PCR. PCR products were analyzed by electrophoresis in a 2% agarose gel. ( D ) Quantitation of the relative level of RNAs from three independent experiments performed as in (C). GFP mRNA was used for normalization. 0.1 μg of the pCI-FLAG-Rev sample (lane 1) was set to 1. Averages and standard deviations are shown. ( E ) Protein from the cell pellets in (C) was analyzed by SDS-PAGE and western blotting with rabbit anti-Gag p55 antiserum or the anti-GFP antibody. UT: untransfected cells were used as a control. U2AF 65 was a loading control. ( F ) The filtrated media from (C) were immunoprecipitated with rabbit anti-Gag p55 antiserum and detected by western blotting with the monoclonal anti-Gag p24 antibody.

Techniques Used: Over Expression, Expressing, Transfection, SDS Page, Western Blot, Plasmid Preparation, Quantitative RT-PCR, Polymerase Chain Reaction, Electrophoresis, Agarose Gel Electrophoresis, Quantitation Assay, Immunoprecipitation

59) Product Images from "A Direct Interaction with NEDD1 Regulates ?-Tubulin Recruitment to the Centrosome"

Article Title: A Direct Interaction with NEDD1 Regulates ?-Tubulin Recruitment to the Centrosome

Journal: PLoS ONE

doi: 10.1371/journal.pone.0009618

NEDD1 residues 572–660 interacts with γ-tubulin directly. (A) The interaction of endogenous NEDD1 and γ-tubulin was assessed in HEK293T cells. Due to its low level of expression, NEDD1 is not detectable in the inputs (1/20 lysates loaded), however γ-tubulin is present (first lane). Both NEDD1 and γ-tubulin are detected in lysates immunoprecipitated with NEDD1 antibody (second lane). NEDD1 is not detected when γ-tubulin is immunoprecipitated (third lane). Additional bands are IgG. In negative controls, γ-tubulin and NEDD1 are not detected when an unrelated antibody (HSP70) (fourth lane) or no antibody (fifth lane) was used for the immunoprecipitation. (B) Full length NEDD1 (660 aa), or two truncation constructs (1–571 and 572–660) were fused to a Myc-tag at their N-terminus. (C) The interaction of full length and truncated forms of Myc-NEDD1 with endogenous γ-tubulin was assessed in HEK293Ts. Expression is confirmed in the inputs (1/20 lysates loaded). γ-tubulin is immunoprecipitated with full length (660 aa) NEDD1, and 572–660 NEDD1, but not 1–571, using a Myc antibody. * represent the correct size for each construct. When no antibody is added (negative controls), no γ-tubulin is immunoprecipitated. (D) The interaction of NEDD1 CTD (residues 572–660) and γ-tubulin was assessed in vitro . Recombinant GST or GST-NEDD1 CTD bound to glutathione sepharose beads were incubated with His-γ-tubulin, with or without HEK293T lysate. Inputs (1/5 lysates loaded) are shown in lanes 6–8. Endogenous γ-tubulin is expressed in the lysate (lane 9). After incubation with the beads and removal of unbound proteins, γ-tubulin is not bound to GST alone (lane 1), but is bound to GST-NEDD1 CTD both in the absence and presence of lysate (lanes 2 and 5 respectively). Endogenous γ-tubulin in the lysate does not to bind to GST alone (lane 3), but does bind to GST-NEDD1 CTD (lane 4).
Figure Legend Snippet: NEDD1 residues 572–660 interacts with γ-tubulin directly. (A) The interaction of endogenous NEDD1 and γ-tubulin was assessed in HEK293T cells. Due to its low level of expression, NEDD1 is not detectable in the inputs (1/20 lysates loaded), however γ-tubulin is present (first lane). Both NEDD1 and γ-tubulin are detected in lysates immunoprecipitated with NEDD1 antibody (second lane). NEDD1 is not detected when γ-tubulin is immunoprecipitated (third lane). Additional bands are IgG. In negative controls, γ-tubulin and NEDD1 are not detected when an unrelated antibody (HSP70) (fourth lane) or no antibody (fifth lane) was used for the immunoprecipitation. (B) Full length NEDD1 (660 aa), or two truncation constructs (1–571 and 572–660) were fused to a Myc-tag at their N-terminus. (C) The interaction of full length and truncated forms of Myc-NEDD1 with endogenous γ-tubulin was assessed in HEK293Ts. Expression is confirmed in the inputs (1/20 lysates loaded). γ-tubulin is immunoprecipitated with full length (660 aa) NEDD1, and 572–660 NEDD1, but not 1–571, using a Myc antibody. * represent the correct size for each construct. When no antibody is added (negative controls), no γ-tubulin is immunoprecipitated. (D) The interaction of NEDD1 CTD (residues 572–660) and γ-tubulin was assessed in vitro . Recombinant GST or GST-NEDD1 CTD bound to glutathione sepharose beads were incubated with His-γ-tubulin, with or without HEK293T lysate. Inputs (1/5 lysates loaded) are shown in lanes 6–8. Endogenous γ-tubulin is expressed in the lysate (lane 9). After incubation with the beads and removal of unbound proteins, γ-tubulin is not bound to GST alone (lane 1), but is bound to GST-NEDD1 CTD both in the absence and presence of lysate (lanes 2 and 5 respectively). Endogenous γ-tubulin in the lysate does not to bind to GST alone (lane 3), but does bind to GST-NEDD1 CTD (lane 4).

Techniques Used: Expressing, Immunoprecipitation, Construct, In Vitro, Recombinant, Incubation

Specific mutations within NEDD1 show reduced binding to γ-tubulin. (A) Mutations were introduced into a Myc-tagged full length NEDD1 construct and transfected into HEK293T cells. The expression of all NEDD1 constructs and γ-tubulin is confirmed in the inputs (1/20 lysates loaded). γ-tubulin is immunoprecipitated with wild type (WT) full length NEDD1 but not with NEDD1 (1–571 aa). There is a loss of γ-tubulin immunoprecipitated with the L642Q mutant NEDD1, but not with the single NEDD1 mutants of L649Q or L656Q. Double mutants including L642Q also reduce the immunoprecipitation of γ-tubulin, as does the L642Q/L649Q/L656Q (3xLQ) triple mutant. The upper band in the γ-tubulin immunoblot is IgG. (B) The interaction of WT or mutant NEDD1 CTD with γ-tubulin was assessed in vitro . Recombinant GST or GST-NEDD1 CTD mutants bound to glutathione sepharose beads were incubated with His-γ-tubulin. All GST-tagged proteins are expressed well and bind to the beads, and His-γ-tubulin is also expressed. After incubation with the beads and removal of unbound proteins, γ-tubulin is not bound to GST alone, but is bound to WT GST-NEDD1 CTD. There is reduced binding of γ-tubulin to L642Q NEDD1, and to the double and triple mutants. The lower bands in the GST-NEDD1 CTD mutant protein lanes are likely to represent cleaved GST. (C) Selected mutations were introduced into a GFP-tagged NEDD1 CTD construct and transfected into HEK293T cells. Expression of all NEDD1 constructs and γ-tubulin is confirmed in the inputs (1/20 lysates loaded). γ-tubulin is immunoprecipitated with full length (1–660 aa) NEDD1 but not with NEDD1 (1–571 aa). WT NEDD1 CTD is able to immunoprecipitate γ-tubulin, as seen previously, as is the E634A/R635A mutant. There is a loss of γ-tubulin immunoprecipitated with the Y636A/S637A and N639A/E640A mutant constructs of NEDD1. The lane indicating - control has no antibody added.
Figure Legend Snippet: Specific mutations within NEDD1 show reduced binding to γ-tubulin. (A) Mutations were introduced into a Myc-tagged full length NEDD1 construct and transfected into HEK293T cells. The expression of all NEDD1 constructs and γ-tubulin is confirmed in the inputs (1/20 lysates loaded). γ-tubulin is immunoprecipitated with wild type (WT) full length NEDD1 but not with NEDD1 (1–571 aa). There is a loss of γ-tubulin immunoprecipitated with the L642Q mutant NEDD1, but not with the single NEDD1 mutants of L649Q or L656Q. Double mutants including L642Q also reduce the immunoprecipitation of γ-tubulin, as does the L642Q/L649Q/L656Q (3xLQ) triple mutant. The upper band in the γ-tubulin immunoblot is IgG. (B) The interaction of WT or mutant NEDD1 CTD with γ-tubulin was assessed in vitro . Recombinant GST or GST-NEDD1 CTD mutants bound to glutathione sepharose beads were incubated with His-γ-tubulin. All GST-tagged proteins are expressed well and bind to the beads, and His-γ-tubulin is also expressed. After incubation with the beads and removal of unbound proteins, γ-tubulin is not bound to GST alone, but is bound to WT GST-NEDD1 CTD. There is reduced binding of γ-tubulin to L642Q NEDD1, and to the double and triple mutants. The lower bands in the GST-NEDD1 CTD mutant protein lanes are likely to represent cleaved GST. (C) Selected mutations were introduced into a GFP-tagged NEDD1 CTD construct and transfected into HEK293T cells. Expression of all NEDD1 constructs and γ-tubulin is confirmed in the inputs (1/20 lysates loaded). γ-tubulin is immunoprecipitated with full length (1–660 aa) NEDD1 but not with NEDD1 (1–571 aa). WT NEDD1 CTD is able to immunoprecipitate γ-tubulin, as seen previously, as is the E634A/R635A mutant. There is a loss of γ-tubulin immunoprecipitated with the Y636A/S637A and N639A/E640A mutant constructs of NEDD1. The lane indicating - control has no antibody added.

Techniques Used: Binding Assay, Construct, Transfection, Expressing, Immunoprecipitation, Mutagenesis, In Vitro, Recombinant, Incubation

60) Product Images from "YB-1 promotes strand separation in vitro of duplex DNA containing either mispaired bases or cisplatin modifications, exhibits endonucleolytic activities and binds several DNA repair proteins"

Article Title: YB-1 promotes strand separation in vitro of duplex DNA containing either mispaired bases or cisplatin modifications, exhibits endonucleolytic activities and binds several DNA repair proteins

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkh170

Immunoblots against DNA repair proteins bound to GST–YB-1 affinity Sepahrose beads. Human 293 embryonic kidney whole cell extracts (WCE) were incubated with either 50 µg of GST–YB-1 or GST linked glutathione–Sepharose
Figure Legend Snippet: Immunoblots against DNA repair proteins bound to GST–YB-1 affinity Sepahrose beads. Human 293 embryonic kidney whole cell extracts (WCE) were incubated with either 50 µg of GST–YB-1 or GST linked glutathione–Sepharose

Techniques Used: Western Blot, Incubation

Characterization of purified human YB-1 on gels stained by Coomassie. (Top, left) This gel contains GST–YB-1 and GST proteins purified on glutathione–Sepharose beads. (Top, right) GST–YB-1 was treated with thrombin to remove the
Figure Legend Snippet: Characterization of purified human YB-1 on gels stained by Coomassie. (Top, left) This gel contains GST–YB-1 and GST proteins purified on glutathione–Sepharose beads. (Top, right) GST–YB-1 was treated with thrombin to remove the

Techniques Used: Purification, Staining

61) Product Images from "Intercalated Disc Protein, mXin?, Suppresses p120-Catenin-Induced Branching Phenotype via Its Interactions with p120-Catenin and Cortactin"

Article Title: Intercalated Disc Protein, mXin?, Suppresses p120-Catenin-Induced Branching Phenotype via Its Interactions with p120-Catenin and Cortactin

Journal: Archives of biochemistry and biophysics

doi: 10.1016/j.abb.2012.12.018

Pull-down assays from heart lysates. Glutathione Sepharose beads pre-bound with various GST-mXinα fragments were used to pull down associated proteins from total heart lysates. After washing the beads, the bound proteins were eluted from beads
Figure Legend Snippet: Pull-down assays from heart lysates. Glutathione Sepharose beads pre-bound with various GST-mXinα fragments were used to pull down associated proteins from total heart lysates. After washing the beads, the bound proteins were eluted from beads

Techniques Used:

Recombinant GST-mXinα directly binds to His-p120-catenin. The pull-down assay was performed with glutathione-Sepharose beads to pull down GST-containing proteins and its interacting proteins from a mixture of purified His-p120-catenin (90 nM)
Figure Legend Snippet: Recombinant GST-mXinα directly binds to His-p120-catenin. The pull-down assay was performed with glutathione-Sepharose beads to pull down GST-containing proteins and its interacting proteins from a mixture of purified His-p120-catenin (90 nM)

Techniques Used: Recombinant, Pull Down Assay, Purification

62) Product Images from "The nucleolar protein Esf2 interacts directly with the DExD/H box RNA helicase, Dbp8, to stimulate ATP hydrolysis"

Article Title: The nucleolar protein Esf2 interacts directly with the DExD/H box RNA helicase, Dbp8, to stimulate ATP hydrolysis

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkl419

Dbp8 directly interacts with the RNA binding protein Esf2 in vivo and in vitro . ( A ) Dbp8 interacts with Esf2 in a yeast 2-hybrid assay. The yeast 2-hybrid host strain carrying the Esf2 bait vector and either Dbp8 prey vector or empty prey vector were serial diluted and tested for growth on permissive (+His) or selective media (−His selection). ( B ) Recombinant Dbp8 directly binds GST-Esf2 in GST pull-down assays. His6-Dbp8 was mixed with equimolar amounts of GST, GST-Esf2 or GST-Rpa34 and incubated on ice for 1 h. As an additional negative control, a GST pull-down assay was performed with GST-Esf2 and His6-Fap7 ( 24 ). GST-fusion proteins were precipitated using glutathione–Sepharose beads and bound proteins (‘P’; lanes 2, 5, 8 and 11) were resolved by SDS–PAGE and stained with Coomassie brilliant blue. Ten percent of the input material (‘I’, lanes 1, 4, 7 and 10) and 10% of the supernatants (‘S’, lanes 3, 6, 9 and 12) was also analyzed. ( C ) Esf2 associates with Dbp8 in vivo . Strains expressing various TAP and/or 3HA-tagged proteins (indicated on top by + or − signs) were grown in YP media to exponential phase. Extracts prepared from these strains were incubated with IgG beads for 1 h at 4°C. Immunoprecipitated proteins were separated by 10% SDS–PAGE and 3HA-tagged proteins were detected by western blot using mouse monoclonal anti-HA antibodies (12CA5; lanes 2, 5 and 6). As a positive control, immunoprecipitations were performed with IgG beads using a strain in which two pre-66S associated proteins were tagged (Rpf1-TAP and Rpf2-3HA; lane 2). As a negative control, a strain was used in which only Esf2 was 3HA-tagged (lane 5). Five percent of the amount of extract used for the immunoprecipitation was also analyzed (lanes 1, 3 and 4). The asterisk indicates a yeast protein that is non-specifically recognized by the anti-HA antibody. ( D ) GST-Esf2 directly binds RNA in vitro . GST-Esf2 or GST alone were incubated with various radiolabeled in vitro transcribed rRNA fragments that contained 5′ETS, 18S or 25S sequences (as illustrated). Complexes were precipitated using glutathione–Sepharose beads and bound RNAs were resolved by 8% denaturing PAGE and visualized by autoradiography (lanes 2 and 5). Ten percent of the input material (‘I’, lanes 1 and 4) and 10% of the supernatants (‘S’, lanes 3 and 6) were also analyzed.
Figure Legend Snippet: Dbp8 directly interacts with the RNA binding protein Esf2 in vivo and in vitro . ( A ) Dbp8 interacts with Esf2 in a yeast 2-hybrid assay. The yeast 2-hybrid host strain carrying the Esf2 bait vector and either Dbp8 prey vector or empty prey vector were serial diluted and tested for growth on permissive (+His) or selective media (−His selection). ( B ) Recombinant Dbp8 directly binds GST-Esf2 in GST pull-down assays. His6-Dbp8 was mixed with equimolar amounts of GST, GST-Esf2 or GST-Rpa34 and incubated on ice for 1 h. As an additional negative control, a GST pull-down assay was performed with GST-Esf2 and His6-Fap7 ( 24 ). GST-fusion proteins were precipitated using glutathione–Sepharose beads and bound proteins (‘P’; lanes 2, 5, 8 and 11) were resolved by SDS–PAGE and stained with Coomassie brilliant blue. Ten percent of the input material (‘I’, lanes 1, 4, 7 and 10) and 10% of the supernatants (‘S’, lanes 3, 6, 9 and 12) was also analyzed. ( C ) Esf2 associates with Dbp8 in vivo . Strains expressing various TAP and/or 3HA-tagged proteins (indicated on top by + or − signs) were grown in YP media to exponential phase. Extracts prepared from these strains were incubated with IgG beads for 1 h at 4°C. Immunoprecipitated proteins were separated by 10% SDS–PAGE and 3HA-tagged proteins were detected by western blot using mouse monoclonal anti-HA antibodies (12CA5; lanes 2, 5 and 6). As a positive control, immunoprecipitations were performed with IgG beads using a strain in which two pre-66S associated proteins were tagged (Rpf1-TAP and Rpf2-3HA; lane 2). As a negative control, a strain was used in which only Esf2 was 3HA-tagged (lane 5). Five percent of the amount of extract used for the immunoprecipitation was also analyzed (lanes 1, 3 and 4). The asterisk indicates a yeast protein that is non-specifically recognized by the anti-HA antibody. ( D ) GST-Esf2 directly binds RNA in vitro . GST-Esf2 or GST alone were incubated with various radiolabeled in vitro transcribed rRNA fragments that contained 5′ETS, 18S or 25S sequences (as illustrated). Complexes were precipitated using glutathione–Sepharose beads and bound RNAs were resolved by 8% denaturing PAGE and visualized by autoradiography (lanes 2 and 5). Ten percent of the input material (‘I’, lanes 1 and 4) and 10% of the supernatants (‘S’, lanes 3 and 6) were also analyzed.

Techniques Used: RNA Binding Assay, In Vivo, In Vitro, Y2H Assay, Plasmid Preparation, Selection, Recombinant, Incubation, Negative Control, Pull Down Assay, SDS Page, Staining, Expressing, Immunoprecipitation, Western Blot, Positive Control, Polyacrylamide Gel Electrophoresis, Autoradiography

Purified recombinant Dbp8 has ATPase activity. ( A ) Purification of His6-Dbp8. Extracts prepared from E.coli expressing His6-Dbp8 (lane 3) was fractionated on a SP Sepharose cation exchange column. The column was extensively washed (lane 5) and proteins were eluted by applying a linear salt gradient. Fractions containing His6-Dbp8 were pooled (lane 6) and His6-Dbp8 was purified from these fractions to near homogeneity using Ni-NTA beads (lane 7). ( B–D ) Optimizing the temperature, pH and salt concentration for Dbp8 ATPase activity. ATP hydrolysis assays were performed at various temperatures (B); 25, 30, 37 and 42°C), different pH (C) and varying potassium chloride concentration (D) with 10 µM ATP. ATP conversion ( Y -axis) was calculated after 30 min by quantifying the phosphate release. Plotted are the averages and standard errors that were derived from three independent experiments.
Figure Legend Snippet: Purified recombinant Dbp8 has ATPase activity. ( A ) Purification of His6-Dbp8. Extracts prepared from E.coli expressing His6-Dbp8 (lane 3) was fractionated on a SP Sepharose cation exchange column. The column was extensively washed (lane 5) and proteins were eluted by applying a linear salt gradient. Fractions containing His6-Dbp8 were pooled (lane 6) and His6-Dbp8 was purified from these fractions to near homogeneity using Ni-NTA beads (lane 7). ( B–D ) Optimizing the temperature, pH and salt concentration for Dbp8 ATPase activity. ATP hydrolysis assays were performed at various temperatures (B); 25, 30, 37 and 42°C), different pH (C) and varying potassium chloride concentration (D) with 10 µM ATP. ATP conversion ( Y -axis) was calculated after 30 min by quantifying the phosphate release. Plotted are the averages and standard errors that were derived from three independent experiments.

Techniques Used: Purification, Recombinant, Activity Assay, Expressing, Concentration Assay, Derivative Assay

63) Product Images from "The serine/arginine-rich protein SF2/ASF regulates protein sumoylation"

Article Title: The serine/arginine-rich protein SF2/ASF regulates protein sumoylation

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1004653107

SF2/ASF interacts with Ubc9 and promotes Topo I and p53 sumoylation in vitro and in living cells. ( A . Cleared lysates were incubated with 2 μg GST or GST-Ubc9 and pulled down with glutathione Sepharose beads. After SDS/PAGE, SF2/ASF binding was analyzed by Western blotting with an anti-T7 antibody. ( B ) In vitro sumoylation reactions were performed using GST-Topoisomerase I (residues 1–200, “Topo I”) as a substrate. Topo I (1 μg, ∼1 μM) was incubated with 150 ng E1 (∼65 nM), 30 ng Ubc9 (∼85 nM), and 1 μg SUMO1 (∼4.5 μM), either with or without purified T7-SF2/ASF (200 ng, ∼360 nM), GST-RanBP2ΔFG (10 ng, ∼8 nM), or GST-Topors (268/644) (400 ng, ∼300 nM) for 30 min. Reactions were stopped by addition of 1 vol of Laemmli sample buffer. One-fourth of the reaction was run in SDS/PAGE and analyzed by Western blot as indicated at the bottom of each panel. ( C ) HEK 293T cells were transfected either with control (Ctl) or SF2/ASF-specific siRNA (15 nM) and, 24 h later, transfected with full-length Myc-tagged Topo I (500 ng) and GFP-SUMO1 (500 ng). Cells were lysed 48 h later in Laemmli sample buffer and subject to Western blot, as indicated at the bottom of each panel. ( D ). Aliquots were taken from the reaction at the indicated time points and analyzed by SDS/PAGE, followed by Western blotting with an anti-p53 antibody. ( E ) Depletion of SF2/ASF affects p53 sumoylation in vivo. HEK 293T cells were transfected either with control (Ctl) or SF2/ASF-specific siRNA (15 nM). Cells were lysed 72 h later in Laemmli sample buffer and subject to Western blot, as indicated at the bottom of each panel.
Figure Legend Snippet: SF2/ASF interacts with Ubc9 and promotes Topo I and p53 sumoylation in vitro and in living cells. ( A . Cleared lysates were incubated with 2 μg GST or GST-Ubc9 and pulled down with glutathione Sepharose beads. After SDS/PAGE, SF2/ASF binding was analyzed by Western blotting with an anti-T7 antibody. ( B ) In vitro sumoylation reactions were performed using GST-Topoisomerase I (residues 1–200, “Topo I”) as a substrate. Topo I (1 μg, ∼1 μM) was incubated with 150 ng E1 (∼65 nM), 30 ng Ubc9 (∼85 nM), and 1 μg SUMO1 (∼4.5 μM), either with or without purified T7-SF2/ASF (200 ng, ∼360 nM), GST-RanBP2ΔFG (10 ng, ∼8 nM), or GST-Topors (268/644) (400 ng, ∼300 nM) for 30 min. Reactions were stopped by addition of 1 vol of Laemmli sample buffer. One-fourth of the reaction was run in SDS/PAGE and analyzed by Western blot as indicated at the bottom of each panel. ( C ) HEK 293T cells were transfected either with control (Ctl) or SF2/ASF-specific siRNA (15 nM) and, 24 h later, transfected with full-length Myc-tagged Topo I (500 ng) and GFP-SUMO1 (500 ng). Cells were lysed 48 h later in Laemmli sample buffer and subject to Western blot, as indicated at the bottom of each panel. ( D ). Aliquots were taken from the reaction at the indicated time points and analyzed by SDS/PAGE, followed by Western blotting with an anti-p53 antibody. ( E ) Depletion of SF2/ASF affects p53 sumoylation in vivo. HEK 293T cells were transfected either with control (Ctl) or SF2/ASF-specific siRNA (15 nM). Cells were lysed 72 h later in Laemmli sample buffer and subject to Western blot, as indicated at the bottom of each panel.

Techniques Used: In Vitro, Incubation, SDS Page, Binding Assay, Western Blot, Purification, Transfection, CTL Assay, In Vivo

SF2/ASF participates in stress-induced sumoylation and regulates Sam68 sumoylation in living cells. ( A ) HEK 293T cells were transfected with the indicated siRNAs. After 24 h, cells were retransfected in every case with an HA-SUMO3 plasmid (500 ng) and with a T7-SF2/ASF ΔRRM2 plasmid (1 μg), when indicated. Forty-eight hours later, cells were exposed to heat shock (42 °C for 15 min) or left untreated, lysed in Laemmli sample buffer, and subject to Western blot as indicated at the bottom of each panel. ( B and C . Cells were then fixed, permeabilized, and incubated with a Sam68 antibody as a nSB marker. Alexa 637-conjugated secondary antibody was used. Sam68 is shown in red and GFP-SUMO in green. (Scale bars, 20 μm.) ( D ) HEK 293T cells were transfected with the indicated siRNAs and 24 h later with the indicated plasmids. After 48 h, cells were harvested and lysates were subject to Ni-NTA agarose purification under denaturing conditions. His-tagged sumoylated proteins were subject to Western blot using an anti-HA antibody. A fraction of each cell lysate (3%) was run in parallel as input control.
Figure Legend Snippet: SF2/ASF participates in stress-induced sumoylation and regulates Sam68 sumoylation in living cells. ( A ) HEK 293T cells were transfected with the indicated siRNAs. After 24 h, cells were retransfected in every case with an HA-SUMO3 plasmid (500 ng) and with a T7-SF2/ASF ΔRRM2 plasmid (1 μg), when indicated. Forty-eight hours later, cells were exposed to heat shock (42 °C for 15 min) or left untreated, lysed in Laemmli sample buffer, and subject to Western blot as indicated at the bottom of each panel. ( B and C . Cells were then fixed, permeabilized, and incubated with a Sam68 antibody as a nSB marker. Alexa 637-conjugated secondary antibody was used. Sam68 is shown in red and GFP-SUMO in green. (Scale bars, 20 μm.) ( D ) HEK 293T cells were transfected with the indicated siRNAs and 24 h later with the indicated plasmids. After 48 h, cells were harvested and lysates were subject to Ni-NTA agarose purification under denaturing conditions. His-tagged sumoylated proteins were subject to Western blot using an anti-HA antibody. A fraction of each cell lysate (3%) was run in parallel as input control.

Techniques Used: Transfection, Plasmid Preparation, Western Blot, Incubation, Marker, Purification

64) Product Images from "The nucleolar protein Esf2 interacts directly with the DExD/H box RNA helicase, Dbp8, to stimulate ATP hydrolysis"

Article Title: The nucleolar protein Esf2 interacts directly with the DExD/H box RNA helicase, Dbp8, to stimulate ATP hydrolysis

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkl419

Dbp8 directly interacts with the RNA binding protein Esf2 in vivo and in vitro . ( A ) Dbp8 interacts with Esf2 in a yeast 2-hybrid assay. The yeast 2-hybrid host strain carrying the Esf2 bait vector and either Dbp8 prey vector or empty prey vector were serial diluted and tested for growth on permissive (+His) or selective media (−His selection). ( B ) Recombinant Dbp8 directly binds GST-Esf2 in GST pull-down assays. His6-Dbp8 was mixed with equimolar amounts of GST, GST-Esf2 or GST-Rpa34 and incubated on ice for 1 h. As an additional negative control, a GST pull-down assay was performed with GST-Esf2 and His6-Fap7 ( 24 ). GST-fusion proteins were precipitated using glutathione–Sepharose beads and bound proteins (‘P’; lanes 2, 5, 8 and 11) were resolved by SDS–PAGE and stained with Coomassie brilliant blue. Ten percent of the input material (‘I’, lanes 1, 4, 7 and 10) and 10% of the supernatants (‘S’, lanes 3, 6, 9 and 12) was also analyzed. ( C ) Esf2 associates with Dbp8 in vivo . Strains expressing various TAP and/or 3HA-tagged proteins (indicated on top by + or − signs) were grown in YP media to exponential phase. Extracts prepared from these strains were incubated with IgG beads for 1 h at 4°C. Immunoprecipitated proteins were separated by 10% SDS–PAGE and 3HA-tagged proteins were detected by western blot using mouse monoclonal anti-HA antibodies (12CA5; lanes 2, 5 and 6). As a positive control, immunoprecipitations were performed with IgG beads using a strain in which two pre-66S associated proteins were tagged (Rpf1-TAP and Rpf2-3HA; lane 2). As a negative control, a strain was used in which only Esf2 was 3HA-tagged (lane 5). Five percent of the amount of extract used for the immunoprecipitation was also analyzed (lanes 1, 3 and 4). The asterisk indicates a yeast protein that is non-specifically recognized by the anti-HA antibody. ( D ) GST-Esf2 directly binds RNA in vitro . GST-Esf2 or GST alone were incubated with various radiolabeled in vitro transcribed rRNA fragments that contained 5′ETS, 18S or 25S sequences (as illustrated). Complexes were precipitated using glutathione–Sepharose beads and bound RNAs were resolved by 8% denaturing PAGE and visualized by autoradiography (lanes 2 and 5). Ten percent of the input material (‘I’, lanes 1 and 4) and 10% of the supernatants (‘S’, lanes 3 and 6) were also analyzed.
Figure Legend Snippet: Dbp8 directly interacts with the RNA binding protein Esf2 in vivo and in vitro . ( A ) Dbp8 interacts with Esf2 in a yeast 2-hybrid assay. The yeast 2-hybrid host strain carrying the Esf2 bait vector and either Dbp8 prey vector or empty prey vector were serial diluted and tested for growth on permissive (+His) or selective media (−His selection). ( B ) Recombinant Dbp8 directly binds GST-Esf2 in GST pull-down assays. His6-Dbp8 was mixed with equimolar amounts of GST, GST-Esf2 or GST-Rpa34 and incubated on ice for 1 h. As an additional negative control, a GST pull-down assay was performed with GST-Esf2 and His6-Fap7 ( 24 ). GST-fusion proteins were precipitated using glutathione–Sepharose beads and bound proteins (‘P’; lanes 2, 5, 8 and 11) were resolved by SDS–PAGE and stained with Coomassie brilliant blue. Ten percent of the input material (‘I’, lanes 1, 4, 7 and 10) and 10% of the supernatants (‘S’, lanes 3, 6, 9 and 12) was also analyzed. ( C ) Esf2 associates with Dbp8 in vivo . Strains expressing various TAP and/or 3HA-tagged proteins (indicated on top by + or − signs) were grown in YP media to exponential phase. Extracts prepared from these strains were incubated with IgG beads for 1 h at 4°C. Immunoprecipitated proteins were separated by 10% SDS–PAGE and 3HA-tagged proteins were detected by western blot using mouse monoclonal anti-HA antibodies (12CA5; lanes 2, 5 and 6). As a positive control, immunoprecipitations were performed with IgG beads using a strain in which two pre-66S associated proteins were tagged (Rpf1-TAP and Rpf2-3HA; lane 2). As a negative control, a strain was used in which only Esf2 was 3HA-tagged (lane 5). Five percent of the amount of extract used for the immunoprecipitation was also analyzed (lanes 1, 3 and 4). The asterisk indicates a yeast protein that is non-specifically recognized by the anti-HA antibody. ( D ) GST-Esf2 directly binds RNA in vitro . GST-Esf2 or GST alone were incubated with various radiolabeled in vitro transcribed rRNA fragments that contained 5′ETS, 18S or 25S sequences (as illustrated). Complexes were precipitated using glutathione–Sepharose beads and bound RNAs were resolved by 8% denaturing PAGE and visualized by autoradiography (lanes 2 and 5). Ten percent of the input material (‘I’, lanes 1 and 4) and 10% of the supernatants (‘S’, lanes 3 and 6) were also analyzed.

Techniques Used: RNA Binding Assay, In Vivo, In Vitro, Y2H Assay, Plasmid Preparation, Selection, Recombinant, Incubation, Negative Control, Pull Down Assay, SDS Page, Staining, Expressing, Immunoprecipitation, Western Blot, Positive Control, Polyacrylamide Gel Electrophoresis, Autoradiography

Purified recombinant Dbp8 has ATPase activity. ( A ) Purification of His6-Dbp8. Extracts prepared from E.coli expressing His6-Dbp8 (lane 3) was fractionated on a SP Sepharose cation exchange column. The column was extensively washed (lane 5) and proteins were eluted by applying a linear salt gradient. Fractions containing His6-Dbp8 were pooled (lane 6) and His6-Dbp8 was purified from these fractions to near homogeneity using Ni-NTA beads (lane 7). ( B–D ) Optimizing the temperature, pH and salt concentration for Dbp8 ATPase activity. ATP hydrolysis assays were performed at various temperatures (B); 25, 30, 37 and 42°C), different pH (C) and varying potassium chloride concentration (D) with 10 µM ATP. ATP conversion ( Y -axis) was calculated after 30 min by quantifying the phosphate release. Plotted are the averages and standard errors that were derived from three independent experiments.
Figure Legend Snippet: Purified recombinant Dbp8 has ATPase activity. ( A ) Purification of His6-Dbp8. Extracts prepared from E.coli expressing His6-Dbp8 (lane 3) was fractionated on a SP Sepharose cation exchange column. The column was extensively washed (lane 5) and proteins were eluted by applying a linear salt gradient. Fractions containing His6-Dbp8 were pooled (lane 6) and His6-Dbp8 was purified from these fractions to near homogeneity using Ni-NTA beads (lane 7). ( B–D ) Optimizing the temperature, pH and salt concentration for Dbp8 ATPase activity. ATP hydrolysis assays were performed at various temperatures (B); 25, 30, 37 and 42°C), different pH (C) and varying potassium chloride concentration (D) with 10 µM ATP. ATP conversion ( Y -axis) was calculated after 30 min by quantifying the phosphate release. Plotted are the averages and standard errors that were derived from three independent experiments.

Techniques Used: Purification, Recombinant, Activity Assay, Expressing, Concentration Assay, Derivative Assay

65) Product Images from "The nucleolar protein Esf2 interacts directly with the DExD/H box RNA helicase, Dbp8, to stimulate ATP hydrolysis"

Article Title: The nucleolar protein Esf2 interacts directly with the DExD/H box RNA helicase, Dbp8, to stimulate ATP hydrolysis

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkl419

Dbp8 directly interacts with the RNA binding protein Esf2 in vivo and in vitro . ( A ) Dbp8 interacts with Esf2 in a yeast 2-hybrid assay. The yeast 2-hybrid host strain carrying the Esf2 bait vector and either Dbp8 prey vector or empty prey vector were serial diluted and tested for growth on permissive (+His) or selective media (−His selection). ( B ) Recombinant Dbp8 directly binds GST-Esf2 in GST pull-down assays. His6-Dbp8 was mixed with equimolar amounts of GST, GST-Esf2 or GST-Rpa34 and incubated on ice for 1 h. As an additional negative control, a GST pull-down assay was performed with GST-Esf2 and His6-Fap7 ( 24 ). GST-fusion proteins were precipitated using glutathione–Sepharose beads and bound proteins (‘P’; lanes 2, 5, 8 and 11) were resolved by SDS–PAGE and stained with Coomassie brilliant blue. Ten percent of the input material (‘I’, lanes 1, 4, 7 and 10) and 10% of the supernatants (‘S’, lanes 3, 6, 9 and 12) was also analyzed. ( C ) Esf2 associates with Dbp8 in vivo . Strains expressing various TAP and/or 3HA-tagged proteins (indicated on top by + or − signs) were grown in YP media to exponential phase. Extracts prepared from these strains were incubated with IgG beads for 1 h at 4°C. Immunoprecipitated proteins were separated by 10% SDS–PAGE and 3HA-tagged proteins were detected by western blot using mouse monoclonal anti-HA antibodies (12CA5; lanes 2, 5 and 6). As a positive control, immunoprecipitations were performed with IgG beads using a strain in which two pre-66S associated proteins were tagged (Rpf1-TAP and Rpf2-3HA; lane 2). As a negative control, a strain was used in which only Esf2 was 3HA-tagged (lane 5). Five percent of the amount of extract used for the immunoprecipitation was also analyzed (lanes 1, 3 and 4). The asterisk indicates a yeast protein that is non-specifically recognized by the anti-HA antibody. ( D ) GST-Esf2 directly binds RNA in vitro . GST-Esf2 or GST alone were incubated with various radiolabeled in vitro transcribed rRNA fragments that contained 5′ETS, 18S or 25S sequences (as illustrated). Complexes were precipitated using glutathione–Sepharose beads and bound RNAs were resolved by 8% denaturing PAGE and visualized by autoradiography (lanes 2 and 5). Ten percent of the input material (‘I’, lanes 1 and 4) and 10% of the supernatants (‘S’, lanes 3 and 6) were also analyzed.
Figure Legend Snippet: Dbp8 directly interacts with the RNA binding protein Esf2 in vivo and in vitro . ( A ) Dbp8 interacts with Esf2 in a yeast 2-hybrid assay. The yeast 2-hybrid host strain carrying the Esf2 bait vector and either Dbp8 prey vector or empty prey vector were serial diluted and tested for growth on permissive (+His) or selective media (−His selection). ( B ) Recombinant Dbp8 directly binds GST-Esf2 in GST pull-down assays. His6-Dbp8 was mixed with equimolar amounts of GST, GST-Esf2 or GST-Rpa34 and incubated on ice for 1 h. As an additional negative control, a GST pull-down assay was performed with GST-Esf2 and His6-Fap7 ( 24 ). GST-fusion proteins were precipitated using glutathione–Sepharose beads and bound proteins (‘P’; lanes 2, 5, 8 and 11) were resolved by SDS–PAGE and stained with Coomassie brilliant blue. Ten percent of the input material (‘I’, lanes 1, 4, 7 and 10) and 10% of the supernatants (‘S’, lanes 3, 6, 9 and 12) was also analyzed. ( C ) Esf2 associates with Dbp8 in vivo . Strains expressing various TAP and/or 3HA-tagged proteins (indicated on top by + or − signs) were grown in YP media to exponential phase. Extracts prepared from these strains were incubated with IgG beads for 1 h at 4°C. Immunoprecipitated proteins were separated by 10% SDS–PAGE and 3HA-tagged proteins were detected by western blot using mouse monoclonal anti-HA antibodies (12CA5; lanes 2, 5 and 6). As a positive control, immunoprecipitations were performed with IgG beads using a strain in which two pre-66S associated proteins were tagged (Rpf1-TAP and Rpf2-3HA; lane 2). As a negative control, a strain was used in which only Esf2 was 3HA-tagged (lane 5). Five percent of the amount of extract used for the immunoprecipitation was also analyzed (lanes 1, 3 and 4). The asterisk indicates a yeast protein that is non-specifically recognized by the anti-HA antibody. ( D ) GST-Esf2 directly binds RNA in vitro . GST-Esf2 or GST alone were incubated with various radiolabeled in vitro transcribed rRNA fragments that contained 5′ETS, 18S or 25S sequences (as illustrated). Complexes were precipitated using glutathione–Sepharose beads and bound RNAs were resolved by 8% denaturing PAGE and visualized by autoradiography (lanes 2 and 5). Ten percent of the input material (‘I’, lanes 1 and 4) and 10% of the supernatants (‘S’, lanes 3 and 6) were also analyzed.

Techniques Used: RNA Binding Assay, In Vivo, In Vitro, Y2H Assay, Plasmid Preparation, Selection, Recombinant, Incubation, Negative Control, Pull Down Assay, SDS Page, Staining, Expressing, Immunoprecipitation, Western Blot, Positive Control, Polyacrylamide Gel Electrophoresis, Autoradiography

Purified recombinant Dbp8 has ATPase activity. ( A ) Purification of His6-Dbp8. Extracts prepared from E.coli expressing His6-Dbp8 (lane 3) was fractionated on a SP Sepharose cation exchange column. The column was extensively washed (lane 5) and proteins were eluted by applying a linear salt gradient. Fractions containing His6-Dbp8 were pooled (lane 6) and His6-Dbp8 was purified from these fractions to near homogeneity using Ni-NTA beads (lane 7). ( B–D ) Optimizing the temperature, pH and salt concentration for Dbp8 ATPase activity. ATP hydrolysis assays were performed at various temperatures (B); 25, 30, 37 and 42°C), different pH (C) and varying potassium chloride concentration (D) with 10 µM ATP. ATP conversion ( Y -axis) was calculated after 30 min by quantifying the phosphate release. Plotted are the averages and standard errors that were derived from three independent experiments.
Figure Legend Snippet: Purified recombinant Dbp8 has ATPase activity. ( A ) Purification of His6-Dbp8. Extracts prepared from E.coli expressing His6-Dbp8 (lane 3) was fractionated on a SP Sepharose cation exchange column. The column was extensively washed (lane 5) and proteins were eluted by applying a linear salt gradient. Fractions containing His6-Dbp8 were pooled (lane 6) and His6-Dbp8 was purified from these fractions to near homogeneity using Ni-NTA beads (lane 7). ( B–D ) Optimizing the temperature, pH and salt concentration for Dbp8 ATPase activity. ATP hydrolysis assays were performed at various temperatures (B); 25, 30, 37 and 42°C), different pH (C) and varying potassium chloride concentration (D) with 10 µM ATP. ATP conversion ( Y -axis) was calculated after 30 min by quantifying the phosphate release. Plotted are the averages and standard errors that were derived from three independent experiments.

Techniques Used: Purification, Recombinant, Activity Assay, Expressing, Concentration Assay, Derivative Assay

66) Product Images from "The BEACH-containing protein WDR81 coordinates p62 and LC3C to promote aggrephagy"

Article Title: The BEACH-containing protein WDR81 coordinates p62 and LC3C to promote aggrephagy

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201608039

WDR81 interacts with LC3C through two LIRs to facilitate aggrephagy. (A and B) Co-IP of GFP-tagged WDR81 fragments with mCh-LC3C. Individual GFP-WDR81 fragments were coexpressed with mCh-LC3C in HEK293 cells. IPs were performed with mCherry antibody, and precipitated proteins were detected with antibodies to GFP and mCherry. (C) Alignment of canonical and noncanonical LIRs in WDR81 with those in p62, NBR1, FUNDC1, or NDP52. Conserved residues are shown in red. (D) Co-IP of mCh-LC3C with wild-type (WT), LIR, or CLIR mutants of GFP-WDR81(341–650) in HEK293 cells. WDR81 mutations are shown in the box. IPs were performed with mCherry antibody and precipitated proteins were detected with antibodies to GFP and mCherry. (E) Co-IP of mCh-LC3C with WT or the indicated LIR mutants of Flag-WDR81 full-length protein in HEK293 cells. IPs were performed as in A. (F) Pull-down of Flag-WDR81 by GST-LC3C. Purified recombinant GST and GST-LC3C immobilized on glutathione-Sepharose beads were incubated overnight at 4°C with lysates of HEK293 cells expressing Flag-WDR81 or the indicated Flag-WDR81 mutants. After extensive washing, bound proteins were subjected to Western blot with Flag antibody. (G and H) KO-81 HeLa cells were transfected with Flag-WDR81 or the indicated Flag-WDR81 mutants. 48 h later, cells were subjected to immunostaining (G) or immunoblotting (H) with p62 antibody. Quantification (mean ± SEM) of p62 in (G) is shown in the right panel. ***, P
Figure Legend Snippet: WDR81 interacts with LC3C through two LIRs to facilitate aggrephagy. (A and B) Co-IP of GFP-tagged WDR81 fragments with mCh-LC3C. Individual GFP-WDR81 fragments were coexpressed with mCh-LC3C in HEK293 cells. IPs were performed with mCherry antibody, and precipitated proteins were detected with antibodies to GFP and mCherry. (C) Alignment of canonical and noncanonical LIRs in WDR81 with those in p62, NBR1, FUNDC1, or NDP52. Conserved residues are shown in red. (D) Co-IP of mCh-LC3C with wild-type (WT), LIR, or CLIR mutants of GFP-WDR81(341–650) in HEK293 cells. WDR81 mutations are shown in the box. IPs were performed with mCherry antibody and precipitated proteins were detected with antibodies to GFP and mCherry. (E) Co-IP of mCh-LC3C with WT or the indicated LIR mutants of Flag-WDR81 full-length protein in HEK293 cells. IPs were performed as in A. (F) Pull-down of Flag-WDR81 by GST-LC3C. Purified recombinant GST and GST-LC3C immobilized on glutathione-Sepharose beads were incubated overnight at 4°C with lysates of HEK293 cells expressing Flag-WDR81 or the indicated Flag-WDR81 mutants. After extensive washing, bound proteins were subjected to Western blot with Flag antibody. (G and H) KO-81 HeLa cells were transfected with Flag-WDR81 or the indicated Flag-WDR81 mutants. 48 h later, cells were subjected to immunostaining (G) or immunoblotting (H) with p62 antibody. Quantification (mean ± SEM) of p62 in (G) is shown in the right panel. ***, P

Techniques Used: Co-Immunoprecipitation Assay, Purification, Recombinant, Incubation, Expressing, Western Blot, Transfection, Immunostaining

WDR81 interacts with p62. (A) Immunostaining of p62 and WDR81 in wild-type (WT) and ATG5 −/− MEF cells. Insets show a magnified view (1.8×) of the boxed area in the merged images. (B) Images of BFP-WDR81 and GFP-p62 coexpressed in HeLa cells (top row); and images of overexpressed GFP-p62 and immunostained endogenous WDR81 (bottom). Dashed lines indicate the cell outline. (C) Colocalization of BFP-WDR81 with GFP-p62 (green arrows) or mCh-WDR91 (red arrows) in HeLa cells. (D) Colocalization of GFP-tagged WDR81 truncations with mCherry-p62 (mCh-p62) in HeLa cells. Schematic representations of the WDR81 truncations are shown in the top panel; the colocalizations are shown in the bottom panel. (E–G) Co-IP of HA-p62 with Flag-WDR81 (E), of HA-p62 with Flag-WDR81(1–650) (F), and of HA-p62 with Flag-WDR81(WD40) (G) in HEK293 cells. IPs were performed using Flag antibody, and precipitated proteins were detected with the indicated antibodies. (H) Interaction of GST-p62 with Flag-WDR81. Purified GST and GST-p62 (left) immobilized on glutathione-Sepharose beads were incubated for 12–16 h at 4°C with lysates of HEK293 cells expressing Flag-WDR81, Flag-WDR81(1–650), or Flag-WDR81(WD40). After extensive washing, bound proteins were subjected to immunoblotting (IB) with antibodies to Flag (right). (I) In vitro interaction of GST-p62 with 35 S-labeled WDR81. (J) Colocalization of GFP-WDR81(1–650) with mCh-tagged p62 truncations in HeLa cells. Schematic representations of the p62 truncations are shown in the top panel and protein colocalizations are shown in the bottom panel. Bars, 10 µm. H, leucine-rich nuclear export signal; L, LC3-interaction region; PB1, Phox and Bem1p domain; UBA, ubiquitin-associated domain; ZZ, ZZ-type zinc-finger domain. (K) In vitro interaction of GST-p62, GST-p62(PB1) with 35 S-labeled WDR81(1–650). (L) Co-IP of mCh-tagged p62 truncations with Flag-WDR81(1–650) in HEK293 cells. IPs were performed using Flag antibody, and precipitated proteins were detected with antibodies to Flag and mCherry. Bars: (main images) 10 µm; (A, insets) 5 µm.
Figure Legend Snippet: WDR81 interacts with p62. (A) Immunostaining of p62 and WDR81 in wild-type (WT) and ATG5 −/− MEF cells. Insets show a magnified view (1.8×) of the boxed area in the merged images. (B) Images of BFP-WDR81 and GFP-p62 coexpressed in HeLa cells (top row); and images of overexpressed GFP-p62 and immunostained endogenous WDR81 (bottom). Dashed lines indicate the cell outline. (C) Colocalization of BFP-WDR81 with GFP-p62 (green arrows) or mCh-WDR91 (red arrows) in HeLa cells. (D) Colocalization of GFP-tagged WDR81 truncations with mCherry-p62 (mCh-p62) in HeLa cells. Schematic representations of the WDR81 truncations are shown in the top panel; the colocalizations are shown in the bottom panel. (E–G) Co-IP of HA-p62 with Flag-WDR81 (E), of HA-p62 with Flag-WDR81(1–650) (F), and of HA-p62 with Flag-WDR81(WD40) (G) in HEK293 cells. IPs were performed using Flag antibody, and precipitated proteins were detected with the indicated antibodies. (H) Interaction of GST-p62 with Flag-WDR81. Purified GST and GST-p62 (left) immobilized on glutathione-Sepharose beads were incubated for 12–16 h at 4°C with lysates of HEK293 cells expressing Flag-WDR81, Flag-WDR81(1–650), or Flag-WDR81(WD40). After extensive washing, bound proteins were subjected to immunoblotting (IB) with antibodies to Flag (right). (I) In vitro interaction of GST-p62 with 35 S-labeled WDR81. (J) Colocalization of GFP-WDR81(1–650) with mCh-tagged p62 truncations in HeLa cells. Schematic representations of the p62 truncations are shown in the top panel and protein colocalizations are shown in the bottom panel. Bars, 10 µm. H, leucine-rich nuclear export signal; L, LC3-interaction region; PB1, Phox and Bem1p domain; UBA, ubiquitin-associated domain; ZZ, ZZ-type zinc-finger domain. (K) In vitro interaction of GST-p62, GST-p62(PB1) with 35 S-labeled WDR81(1–650). (L) Co-IP of mCh-tagged p62 truncations with Flag-WDR81(1–650) in HEK293 cells. IPs were performed using Flag antibody, and precipitated proteins were detected with antibodies to Flag and mCherry. Bars: (main images) 10 µm; (A, insets) 5 µm.

Techniques Used: Immunostaining, Co-Immunoprecipitation Assay, Purification, Incubation, Expressing, In Vitro, Labeling

67) Product Images from "Lettuce‐produced hepatitis C virus E1E2 heterodimer triggers immune responses in mice and antibody production after oral vaccination"

Article Title: Lettuce‐produced hepatitis C virus E1E2 heterodimer triggers immune responses in mice and antibody production after oral vaccination

Journal: Plant Biotechnology Journal

doi: 10.1111/pbi.12743

Expression of the HCV E1E2‐encoding gene in mammalian cells. (a) Lysates of control (lane 1) or E1E2‐expressing HEK ‐293T cells (lane 2) were subjected to Western blotting followed by sequential detection with anti‐E2 Abs 3/11 and anti‐E1 Abs A4. (b) The GNA ‐Sepharose purified E1E2 heterodimer (lane 1) was twofold serially diluted (lanes 2‐10), followed by detection of the E2 polypeptide by Western blotting using the anti‐E2 Abs 3/11.
Figure Legend Snippet: Expression of the HCV E1E2‐encoding gene in mammalian cells. (a) Lysates of control (lane 1) or E1E2‐expressing HEK ‐293T cells (lane 2) were subjected to Western blotting followed by sequential detection with anti‐E2 Abs 3/11 and anti‐E1 Abs A4. (b) The GNA ‐Sepharose purified E1E2 heterodimer (lane 1) was twofold serially diluted (lanes 2‐10), followed by detection of the E2 polypeptide by Western blotting using the anti‐E2 Abs 3/11.

Techniques Used: Expressing, Western Blot, Purification

CD 81 binding of lettuce‐produced HCV antigens. Protein extracts (input) from HEK 293T cells expressing the E1E2 dimer as control (a) and Lactuca sativa Veronique leaves expressing the E1E2 or E1E2ΔN6 dimers (b) were reacted with GST ‐fused CD 81‐ LEL adsorbed onto glutathione–Sepharose beads. Lysates and CD 81‐bound proteins were analysed by Western blotting and sequentially detected with anti‐E2 3/11 and anti‐E1A4 Abs.
Figure Legend Snippet: CD 81 binding of lettuce‐produced HCV antigens. Protein extracts (input) from HEK 293T cells expressing the E1E2 dimer as control (a) and Lactuca sativa Veronique leaves expressing the E1E2 or E1E2ΔN6 dimers (b) were reacted with GST ‐fused CD 81‐ LEL adsorbed onto glutathione–Sepharose beads. Lysates and CD 81‐bound proteins were analysed by Western blotting and sequentially detected with anti‐E2 3/11 and anti‐E1A4 Abs.

Techniques Used: Binding Assay, Produced, Expressing, Western Blot

68) Product Images from "Downregulation of the proapoptotic protein MOAP-1 by the UBR5 ubiquitin ligase and its role in ovarian cancer resistance to cisplatin"

Article Title: Downregulation of the proapoptotic protein MOAP-1 by the UBR5 ubiquitin ligase and its role in ovarian cancer resistance to cisplatin

Journal: Oncogene

doi: 10.1038/onc.2016.336

UBR5-containing EDVP E3 ligase complex interacts and regulates MOAP-1 ubiquitylation and stability. ( a ) Flag-MOAP-1 was transfected into 293T cells, and lysates were prepared 48 h posttransfection. Co-IP with Flag M2 agarose beads were performed and immunoblotted with antibodies as indicated. n =3 independent experiments. ( b ) In vitro ubiquitylation assay was performed as Figure 2d in the presence or absence of recombinant DDB1, VprBP and Dyrk2. n =3 independent experiments. Original uncropped image is shown in Supplementary Figure S6 . ( c ) siCtrl or siDyrk2 was transfected into H1299 or 293T cells, and Flag-MOAP-1 was transfected into each siRNA transfectant 24 h post-siRNA transfection. Cell lysates were prepared under denaturing condition; Flag-MOAP-1 was immunoprecipitated and immunoblotted with ubiquitin antibody. Same membrane was re-blotted with Flag antibody for Flag-MOAP-1. n =3 independent experiments. Asterisk in Dyrk2 blot indicates non-specific band. Quantification of MOAP-1 ubiquitylation is shown in Supplementary Figure S3B . ( d ) 293T cells transfected with siCtrl, siDyrk2 or siUBR5 were treated with CHX 48 h posttransfection. Cells were collected at the indicated times after CHX treatment, and lysates were prepared and immunoblotted as indicated (left). MOAP-1 protein level was quantified and plotted (right). The MOAP-1 abundance at 0 time point was set at 100%. n =4 independent experiments (means±s.e.m.). Molecular weight markers are in kDa.
Figure Legend Snippet: UBR5-containing EDVP E3 ligase complex interacts and regulates MOAP-1 ubiquitylation and stability. ( a ) Flag-MOAP-1 was transfected into 293T cells, and lysates were prepared 48 h posttransfection. Co-IP with Flag M2 agarose beads were performed and immunoblotted with antibodies as indicated. n =3 independent experiments. ( b ) In vitro ubiquitylation assay was performed as Figure 2d in the presence or absence of recombinant DDB1, VprBP and Dyrk2. n =3 independent experiments. Original uncropped image is shown in Supplementary Figure S6 . ( c ) siCtrl or siDyrk2 was transfected into H1299 or 293T cells, and Flag-MOAP-1 was transfected into each siRNA transfectant 24 h post-siRNA transfection. Cell lysates were prepared under denaturing condition; Flag-MOAP-1 was immunoprecipitated and immunoblotted with ubiquitin antibody. Same membrane was re-blotted with Flag antibody for Flag-MOAP-1. n =3 independent experiments. Asterisk in Dyrk2 blot indicates non-specific band. Quantification of MOAP-1 ubiquitylation is shown in Supplementary Figure S3B . ( d ) 293T cells transfected with siCtrl, siDyrk2 or siUBR5 were treated with CHX 48 h posttransfection. Cells were collected at the indicated times after CHX treatment, and lysates were prepared and immunoblotted as indicated (left). MOAP-1 protein level was quantified and plotted (right). The MOAP-1 abundance at 0 time point was set at 100%. n =4 independent experiments (means±s.e.m.). Molecular weight markers are in kDa.

Techniques Used: Transfection, Co-Immunoprecipitation Assay, In Vitro, Ubiquitin Assay, Recombinant, Immunoprecipitation, Molecular Weight

UBR5 is identified as a novel interacting factor of MOAP-1. ( a ) Flag-MOAP-1 was transfected into 293T cells, treated with or without 100 μ M of etoposide (ETP) for 18 or 24 h and lysates were prepared for co-immunoprecipitation (Co-IP) with Flag M2 agarose beads. Co-IP samples were applied for SDS–PAGE and proteins were visualized by silver staining (top). Whole-cell lysates were immunoblotted with Flag antibody for Flag-MOAP-1 (bottom). n =2 independent experiments. ( b ) Transfection was performed as in panel ( a ), and Co-IP samples with Flag beads were immunoblotted as indicated. n =3 independent experiments. ( c ) GST-MOAP-1 recombinant protein was incubated with or without His-UBR5 recombinant protein on ice for 4 h, and nickel beads were added and incubated for 45 min. Beads were washed with 0.5% TritonX-100 wash buffer for five times. The proteins were immunoblotted with UBR5 or MOAP-1 antibody. n =3 independent experiments. Molecular weight markers are in kDa.
Figure Legend Snippet: UBR5 is identified as a novel interacting factor of MOAP-1. ( a ) Flag-MOAP-1 was transfected into 293T cells, treated with or without 100 μ M of etoposide (ETP) for 18 or 24 h and lysates were prepared for co-immunoprecipitation (Co-IP) with Flag M2 agarose beads. Co-IP samples were applied for SDS–PAGE and proteins were visualized by silver staining (top). Whole-cell lysates were immunoblotted with Flag antibody for Flag-MOAP-1 (bottom). n =2 independent experiments. ( b ) Transfection was performed as in panel ( a ), and Co-IP samples with Flag beads were immunoblotted as indicated. n =3 independent experiments. ( c ) GST-MOAP-1 recombinant protein was incubated with or without His-UBR5 recombinant protein on ice for 4 h, and nickel beads were added and incubated for 45 min. Beads were washed with 0.5% TritonX-100 wash buffer for five times. The proteins were immunoblotted with UBR5 or MOAP-1 antibody. n =3 independent experiments. Molecular weight markers are in kDa.

Techniques Used: Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, SDS Page, Silver Staining, Recombinant, Incubation, Molecular Weight

69) Product Images from "TRIM32-Cytoplasmic-Body Formation Is an ATP-Consuming Process Stimulated by HSP70 in Cells"

Article Title: TRIM32-Cytoplasmic-Body Formation Is an ATP-Consuming Process Stimulated by HSP70 in Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0169436

Identification of HSP70 as a major binding partner of the overexpressed TRIM32 polypeptide. ( A ) Schematic representation of the human TRIM32 protein (upper) and its MEF-fused version (lower). ( B ) Association of TRIM32 and HSP70 as assessed with western blotting (WB). HEK293 cells were transfected with the FLAG-TRIM32 construct or the vector alone, with (lanes 1–3) or without (lanes 4–6) myc-HSP70. The lysates were separated with SDS-PAGE and western-blotted with anti-FLAG (for FLAG-TRIM32), anti-myc (for myc-HSP70), or anti-HSP70 (for endogenous HSP70) (lower panels, marked as “Ext”). FLAG-TRIM32 proteins were also immunoprecipitated from the lysates with nonimmune IgG (c-IgG) or anti-FLAG Sepharose beads (anti-FLAG). The immunoprecipitates were western-blotted with the aforesaid antibodies (upper panels, marked as “IP”). Lane 6′ represents lane 6 at a higher exposure. ( C ) HSP70 directly binds to TRIM32 depending on the specific nucleotide-binding state. Purified GST-TRIM32 or GST alone immobilized on agarose beads was incubated with purified HSP70 (lanes 1–3) or the K71S mutant protein (lanes 4–6) in the presence or absence of ATP or ADP, and then western-blotted with anti-HSP70 (for HSP70 and K71S) or anti-GST (for GST-TRIM32 and GST). “+” and “-” respectively indicate presence and absence of ATP/ADP. ( D ) FLAG-TRIM32-myc-HSP70/K71S-transfected HEK293 cells were co-immunoprecipitated to analyze the association between TRMI32 and K71S, as described in (B).
Figure Legend Snippet: Identification of HSP70 as a major binding partner of the overexpressed TRIM32 polypeptide. ( A ) Schematic representation of the human TRIM32 protein (upper) and its MEF-fused version (lower). ( B ) Association of TRIM32 and HSP70 as assessed with western blotting (WB). HEK293 cells were transfected with the FLAG-TRIM32 construct or the vector alone, with (lanes 1–3) or without (lanes 4–6) myc-HSP70. The lysates were separated with SDS-PAGE and western-blotted with anti-FLAG (for FLAG-TRIM32), anti-myc (for myc-HSP70), or anti-HSP70 (for endogenous HSP70) (lower panels, marked as “Ext”). FLAG-TRIM32 proteins were also immunoprecipitated from the lysates with nonimmune IgG (c-IgG) or anti-FLAG Sepharose beads (anti-FLAG). The immunoprecipitates were western-blotted with the aforesaid antibodies (upper panels, marked as “IP”). Lane 6′ represents lane 6 at a higher exposure. ( C ) HSP70 directly binds to TRIM32 depending on the specific nucleotide-binding state. Purified GST-TRIM32 or GST alone immobilized on agarose beads was incubated with purified HSP70 (lanes 1–3) or the K71S mutant protein (lanes 4–6) in the presence or absence of ATP or ADP, and then western-blotted with anti-HSP70 (for HSP70 and K71S) or anti-GST (for GST-TRIM32 and GST). “+” and “-” respectively indicate presence and absence of ATP/ADP. ( D ) FLAG-TRIM32-myc-HSP70/K71S-transfected HEK293 cells were co-immunoprecipitated to analyze the association between TRMI32 and K71S, as described in (B).

Techniques Used: Binding Assay, Western Blot, Transfection, Construct, Plasmid Preparation, SDS Page, Immunoprecipitation, Purification, Incubation, Mutagenesis

The HSP70-TRIM32 complex is biochemically separate from the 14-3-3-TRIM32 phospho-complex. ( A ) HEK293 cells were transiently transfected with FLAG-TRIM32 or its truncated mutants, immunoprecipitated with anti-FLAG, then western-blotted with anti-HSP70 and anti-FLAG. The 14-3-3 binding data are from our previous study [ 8 ]. “Full,” “C-140,” “C-265,” “C-365,” and “N-361” indicate the full TRIM32 protein and its truncated mutants. ( B ) HEK293 cells were transfected with FLAG-TRIM32, the PKA catalytic subunit, and increasing amounts of myc-14-3-3η (indicated by the shaded triangle above the western blot [WB]). Transfection was followed by immunoprecipitation with anti-FLAG, and then western blots, as described in (A). ( C ) The FLAG-TRIM32 complex shown in lane 5 of (B) was further subjected to immunoprecipitation with anti-myc Sepharose beads to purify the myc-14-3-3-FLAG-TRIM32 complex, and western-blotted with specific anti-HSP70 antibody to monitor endogenous HSP70 presence (left panel). The right panel shows the myc-HSP70-FLAG-TRIM32 complex with a western blot using anti-14-3-3 antibodies to monitor endogenous 14-3-3 presence.
Figure Legend Snippet: The HSP70-TRIM32 complex is biochemically separate from the 14-3-3-TRIM32 phospho-complex. ( A ) HEK293 cells were transiently transfected with FLAG-TRIM32 or its truncated mutants, immunoprecipitated with anti-FLAG, then western-blotted with anti-HSP70 and anti-FLAG. The 14-3-3 binding data are from our previous study [ 8 ]. “Full,” “C-140,” “C-265,” “C-365,” and “N-361” indicate the full TRIM32 protein and its truncated mutants. ( B ) HEK293 cells were transfected with FLAG-TRIM32, the PKA catalytic subunit, and increasing amounts of myc-14-3-3η (indicated by the shaded triangle above the western blot [WB]). Transfection was followed by immunoprecipitation with anti-FLAG, and then western blots, as described in (A). ( C ) The FLAG-TRIM32 complex shown in lane 5 of (B) was further subjected to immunoprecipitation with anti-myc Sepharose beads to purify the myc-14-3-3-FLAG-TRIM32 complex, and western-blotted with specific anti-HSP70 antibody to monitor endogenous HSP70 presence (left panel). The right panel shows the myc-HSP70-FLAG-TRIM32 complex with a western blot using anti-14-3-3 antibodies to monitor endogenous 14-3-3 presence.

Techniques Used: Transfection, Immunoprecipitation, Western Blot, Binding Assay

70) Product Images from "Protein kinase DYRK2 is an E3-ligase specific molecular assembler"

Article Title: Protein kinase DYRK2 is an E3-ligase specific molecular assembler

Journal: Nature cell biology

doi: 10.1038/ncb1848

Katanin p60 is the ubiquitination substrate for EDVP E3 ligase complex (a) Control (IgG) or anti-FLAG immunoprecipitates were prepared from 293T cells transfected with plasmid encoding a triple tagged Katanin. Western blotting was conducted using indicated antibodies to show a specific interaction between the DYRK2-EDVP complex and Katanin p60. (b) Bacterially expressed recombinant MBP-tagged EDD, DDB1 or VPRBP bound to amylose sepharose beads were incubated with recombinant GST-Katanin and the association of Katanin was detected by western blotting with anti-GST antibody. The expression of MBP-fusion proteins was detected by anti-MBP antibody. (c) HeLa cells were transfected with either control siRNA or VPRBP siRNA and the association of EDD and DDB1 with Katanin was assessed by immunoblotting with their respective antibodies after immunoprecipitation using anti-Katanin antibody. (d) HeLa cells were transfected with different siRNAs as indicated. Cell lysates prepared after 5 hour MG132 (10µM) treatment were subjected to immunoprecipitaton using anti-Katanin antibodies. The ubiquitinated Katanin was detected with anti-ubiquitin antibody. The protein expression and the specificity of different siRNAs were confirmed by immunoblotting of cell extracts using antibodies as indicated. (e) HeLa cells transfected with EDD specific siRNA were retransfected with either siRNA resistant wild type EDD (SiR-EDD WT) or catalytically inactive EDD (SiR-EDD C/A). Ubiquitination of Katanin was assessed by immunoblotting with anti-ubiquitin antibody after immunoprecipitating with anti-Katanin antibody. The expression of endogenous EDD and the transfected siRNA resistant EDD was assessed by immunoblotting with anti-EDD antibody.
Figure Legend Snippet: Katanin p60 is the ubiquitination substrate for EDVP E3 ligase complex (a) Control (IgG) or anti-FLAG immunoprecipitates were prepared from 293T cells transfected with plasmid encoding a triple tagged Katanin. Western blotting was conducted using indicated antibodies to show a specific interaction between the DYRK2-EDVP complex and Katanin p60. (b) Bacterially expressed recombinant MBP-tagged EDD, DDB1 or VPRBP bound to amylose sepharose beads were incubated with recombinant GST-Katanin and the association of Katanin was detected by western blotting with anti-GST antibody. The expression of MBP-fusion proteins was detected by anti-MBP antibody. (c) HeLa cells were transfected with either control siRNA or VPRBP siRNA and the association of EDD and DDB1 with Katanin was assessed by immunoblotting with their respective antibodies after immunoprecipitation using anti-Katanin antibody. (d) HeLa cells were transfected with different siRNAs as indicated. Cell lysates prepared after 5 hour MG132 (10µM) treatment were subjected to immunoprecipitaton using anti-Katanin antibodies. The ubiquitinated Katanin was detected with anti-ubiquitin antibody. The protein expression and the specificity of different siRNAs were confirmed by immunoblotting of cell extracts using antibodies as indicated. (e) HeLa cells transfected with EDD specific siRNA were retransfected with either siRNA resistant wild type EDD (SiR-EDD WT) or catalytically inactive EDD (SiR-EDD C/A). Ubiquitination of Katanin was assessed by immunoblotting with anti-ubiquitin antibody after immunoprecipitating with anti-Katanin antibody. The expression of endogenous EDD and the transfected siRNA resistant EDD was assessed by immunoblotting with anti-EDD antibody.

Techniques Used: Transfection, Plasmid Preparation, Western Blot, Recombinant, Incubation, Expressing, Immunoprecipitation

Identification of EDD-DDB1-VPRBP as DYRK2 associated proteins (a) Tandem affinity purification of DYRK2-containg protein complexes was conducted using 293T cells stably expressing triple tagged DYRK2. Associated proteins were separated by SDS-PAGE and visualized by Coomassie staining. The proteins and the number of peptides identified by mass spectrometry analysis are shown in the table on the right and also in supplementary data ( Supplemental Table 1 ) (b) Immunoprecipitation using control IgG or anti-FLAG (DYRK2) antibody were performed using extracts prepared from 293T derivative cells stably expressing FLAG-tagged DYRK2. The presence of EDD, DDB1, VPRBP, Cul4A or Roc1 in these immunoprecipitates was evaluated by immunoblotting with their respective antibodies. (c) Reverse co-immunoprecipitation experiments were performed using anti-EDD, anti-Cul4A, anti-DDB1 and anti-VPRBP antibodies and the associated endogenous DYRK2 and other indicated proteins was identified by Western blotting using their respective antibodies. (d) GST pull down assay was performed using immobilized control GST or GST-DYRK2 fusion proteins on agarose beads and incubated with extracts prepared from 293T cells. The interaction of EDD, DDB1, VPRBP or Cul4A with DYRK2 was assessed by immunoblotting with their respective antibodies.
Figure Legend Snippet: Identification of EDD-DDB1-VPRBP as DYRK2 associated proteins (a) Tandem affinity purification of DYRK2-containg protein complexes was conducted using 293T cells stably expressing triple tagged DYRK2. Associated proteins were separated by SDS-PAGE and visualized by Coomassie staining. The proteins and the number of peptides identified by mass spectrometry analysis are shown in the table on the right and also in supplementary data ( Supplemental Table 1 ) (b) Immunoprecipitation using control IgG or anti-FLAG (DYRK2) antibody were performed using extracts prepared from 293T derivative cells stably expressing FLAG-tagged DYRK2. The presence of EDD, DDB1, VPRBP, Cul4A or Roc1 in these immunoprecipitates was evaluated by immunoblotting with their respective antibodies. (c) Reverse co-immunoprecipitation experiments were performed using anti-EDD, anti-Cul4A, anti-DDB1 and anti-VPRBP antibodies and the associated endogenous DYRK2 and other indicated proteins was identified by Western blotting using their respective antibodies. (d) GST pull down assay was performed using immobilized control GST or GST-DYRK2 fusion proteins on agarose beads and incubated with extracts prepared from 293T cells. The interaction of EDD, DDB1, VPRBP or Cul4A with DYRK2 was assessed by immunoblotting with their respective antibodies.

Techniques Used: Affinity Purification, Stable Transfection, Expressing, SDS Page, Staining, Mass Spectrometry, Immunoprecipitation, Western Blot, Pull Down Assay, Incubation

71) Product Images from "Cotton Leaf Curl Multan virus C4 protein suppresses both transcriptional and post-transcriptional gene silencing by interacting with SAM synthetase"

Article Title: Cotton Leaf Curl Multan virus C4 protein suppresses both transcriptional and post-transcriptional gene silencing by interacting with SAM synthetase

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1007282

CLCuMuV C4 protein interacts with NbSAMS2 in vitro and in vivo . (A) CLCuMuV C4 protein co-immunoprecipitated with NbSAMS2. Total protein extracts were immunoprecipitated with anti-GFP beads and then monitored by immunoblotting (IB) using anti-GFP or anti-HA antibodies. cLUC represents c-terminal fragment of the firefly luciferase. (B) GST pull-down assay showed the interaction between C4 and NbSAMS2. Total soluble proteins of E . coli expressing GST-NbSAMS2 or GST were incubated with C4- His or C4 R13A -His immobilized on glutathione-sepharose beads and monitored by anti-His antibody. (C) BiFC assay showed the interaction between C4 and NbSAMS2. Cells were photographed 60 hpi using confocal laser scanning microscope. Bar scale represents 50 μm. (D) Western blot analyses of BiFC construct combinations from the same experiments as in (C). All combinations were detected with anti-GFP polyclonal antibody.
Figure Legend Snippet: CLCuMuV C4 protein interacts with NbSAMS2 in vitro and in vivo . (A) CLCuMuV C4 protein co-immunoprecipitated with NbSAMS2. Total protein extracts were immunoprecipitated with anti-GFP beads and then monitored by immunoblotting (IB) using anti-GFP or anti-HA antibodies. cLUC represents c-terminal fragment of the firefly luciferase. (B) GST pull-down assay showed the interaction between C4 and NbSAMS2. Total soluble proteins of E . coli expressing GST-NbSAMS2 or GST were incubated with C4- His or C4 R13A -His immobilized on glutathione-sepharose beads and monitored by anti-His antibody. (C) BiFC assay showed the interaction between C4 and NbSAMS2. Cells were photographed 60 hpi using confocal laser scanning microscope. Bar scale represents 50 μm. (D) Western blot analyses of BiFC construct combinations from the same experiments as in (C). All combinations were detected with anti-GFP polyclonal antibody.

Techniques Used: In Vitro, In Vivo, Immunoprecipitation, Luciferase, Pull Down Assay, Expressing, Incubation, Bimolecular Fluorescence Complementation Assay, Laser-Scanning Microscopy, Western Blot, Construct

Silencing of NbSAMS2 enhance plant susceptibility against CLCuMuV infection. (A) Symptom of NbSAMS2 -silenced or control plants at 14 dpi. N . benthamiana plants were co-inoculated with CLCuMuV and its beta satellite VIGS vector containing DNA fragment of NbSAMS2 or GFP . (B) Southern blot analysis of viral DNAs in CLCuMuV-infected plants shown in ( A ). Total DNAs were blotted with biotin-labeled probes specific for CLCuMuV V1. The DNA agarose gel was stained with ethidium bromide as a loading control. Viral single-stranded DNA (ssDNA) and supercoiled DNA (scDNA) are indicated. (C) Silencing of NbSAMS2 increased viral DNA accumulation. Real-time PCR analysis of V1 gene from CLCuMuV was used to determine viral DNA level. Values represent means ± SE from three independent experiments. (*p
Figure Legend Snippet: Silencing of NbSAMS2 enhance plant susceptibility against CLCuMuV infection. (A) Symptom of NbSAMS2 -silenced or control plants at 14 dpi. N . benthamiana plants were co-inoculated with CLCuMuV and its beta satellite VIGS vector containing DNA fragment of NbSAMS2 or GFP . (B) Southern blot analysis of viral DNAs in CLCuMuV-infected plants shown in ( A ). Total DNAs were blotted with biotin-labeled probes specific for CLCuMuV V1. The DNA agarose gel was stained with ethidium bromide as a loading control. Viral single-stranded DNA (ssDNA) and supercoiled DNA (scDNA) are indicated. (C) Silencing of NbSAMS2 increased viral DNA accumulation. Real-time PCR analysis of V1 gene from CLCuMuV was used to determine viral DNA level. Values represent means ± SE from three independent experiments. (*p

Techniques Used: Infection, Plasmid Preparation, Southern Blot, Labeling, Agarose Gel Electrophoresis, Staining, Real-time Polymerase Chain Reaction

72) Product Images from "Cotton Leaf Curl Multan virus C4 protein suppresses both transcriptional and post-transcriptional gene silencing by interacting with SAM synthetase"

Article Title: Cotton Leaf Curl Multan virus C4 protein suppresses both transcriptional and post-transcriptional gene silencing by interacting with SAM synthetase

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1007282

CLCuMuV C4 protein interacts with NbSAMS2 in vitro and in vivo . (A) CLCuMuV C4 protein co-immunoprecipitated with NbSAMS2. Total protein extracts were immunoprecipitated with anti-GFP beads and then monitored by immunoblotting (IB) using anti-GFP or anti-HA antibodies. cLUC represents c-terminal fragment of the firefly luciferase. (B) GST pull-down assay showed the interaction between C4 and NbSAMS2. Total soluble proteins of E . coli expressing GST-NbSAMS2 or GST were incubated with C4- His or C4 R13A -His immobilized on glutathione-sepharose beads and monitored by anti-His antibody. (C) BiFC assay showed the interaction between C4 and NbSAMS2. Cells were photographed 60 hpi using confocal laser scanning microscope. Bar scale represents 50 μm. (D) Western blot analyses of BiFC construct combinations from the same experiments as in (C). All combinations were detected with anti-GFP polyclonal antibody.
Figure Legend Snippet: CLCuMuV C4 protein interacts with NbSAMS2 in vitro and in vivo . (A) CLCuMuV C4 protein co-immunoprecipitated with NbSAMS2. Total protein extracts were immunoprecipitated with anti-GFP beads and then monitored by immunoblotting (IB) using anti-GFP or anti-HA antibodies. cLUC represents c-terminal fragment of the firefly luciferase. (B) GST pull-down assay showed the interaction between C4 and NbSAMS2. Total soluble proteins of E . coli expressing GST-NbSAMS2 or GST were incubated with C4- His or C4 R13A -His immobilized on glutathione-sepharose beads and monitored by anti-His antibody. (C) BiFC assay showed the interaction between C4 and NbSAMS2. Cells were photographed 60 hpi using confocal laser scanning microscope. Bar scale represents 50 μm. (D) Western blot analyses of BiFC construct combinations from the same experiments as in (C). All combinations were detected with anti-GFP polyclonal antibody.

Techniques Used: In Vitro, In Vivo, Immunoprecipitation, Luciferase, Pull Down Assay, Expressing, Incubation, Bimolecular Fluorescence Complementation Assay, Laser-Scanning Microscopy, Western Blot, Construct

Silencing of NbSAMS2 enhance plant susceptibility against CLCuMuV infection. (A) Symptom of NbSAMS2 -silenced or control plants at 14 dpi. N . benthamiana plants were co-inoculated with CLCuMuV and its beta satellite VIGS vector containing DNA fragment of NbSAMS2 or GFP . (B) Southern blot analysis of viral DNAs in CLCuMuV-infected plants shown in ( A ). Total DNAs were blotted with biotin-labeled probes specific for CLCuMuV V1. The DNA agarose gel was stained with ethidium bromide as a loading control. Viral single-stranded DNA (ssDNA) and supercoiled DNA (scDNA) are indicated. (C) Silencing of NbSAMS2 increased viral DNA accumulation. Real-time PCR analysis of V1 gene from CLCuMuV was used to determine viral DNA level. Values represent means ± SE from three independent experiments. (*p
Figure Legend Snippet: Silencing of NbSAMS2 enhance plant susceptibility against CLCuMuV infection. (A) Symptom of NbSAMS2 -silenced or control plants at 14 dpi. N . benthamiana plants were co-inoculated with CLCuMuV and its beta satellite VIGS vector containing DNA fragment of NbSAMS2 or GFP . (B) Southern blot analysis of viral DNAs in CLCuMuV-infected plants shown in ( A ). Total DNAs were blotted with biotin-labeled probes specific for CLCuMuV V1. The DNA agarose gel was stained with ethidium bromide as a loading control. Viral single-stranded DNA (ssDNA) and supercoiled DNA (scDNA) are indicated. (C) Silencing of NbSAMS2 increased viral DNA accumulation. Real-time PCR analysis of V1 gene from CLCuMuV was used to determine viral DNA level. Values represent means ± SE from three independent experiments. (*p

Techniques Used: Infection, Plasmid Preparation, Southern Blot, Labeling, Agarose Gel Electrophoresis, Staining, Real-time Polymerase Chain Reaction

73) Product Images from "Polycomb Group Protein PHF1 Regulates p53-dependent Cell Growth Arrest and Apoptosis *"

Article Title: Polycomb Group Protein PHF1 Regulates p53-dependent Cell Growth Arrest and Apoptosis *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.338996

PHF1 interacts with p53 both in vivo and in vitro . A , H1299 cells were transfected with Myc-PHF1 and/or HA-p53 as indicated. Cell lysates were prepared and subjected to immunoprecipitation ( IP ) with anti-HA or Myc antibody. The immunoprecipitates were detected by Western blot ( WB ) with anti-Myc and anti-HA antibodies, respectively. B , co-immunoprecipitation of endogenous PHF1 and p53 proteins in U2OS cells. The endogenous p53 was immunoprecipitated by p53 (FL-393) antibody with rabbit IgG as a negative control. The immunoprecipitates were detected by Western blot with anti-PHF1 and p53 antibodies, respectively. C , H1299 cells were transfected with Myc-p53 and/or ( .) GFP-PHF1. Cells were fixed 48 h after transfection and subjected to immunofluorescence analysis. D , bacterially expressed GST fusion PHF1 protein bound to glutathione-Sepharose beads and incubated with His-tagged fusion p53 protein. Bound His-p53 was detected by Western blot with anti-His antibody. M.W. , molecular weight.
Figure Legend Snippet: PHF1 interacts with p53 both in vivo and in vitro . A , H1299 cells were transfected with Myc-PHF1 and/or HA-p53 as indicated. Cell lysates were prepared and subjected to immunoprecipitation ( IP ) with anti-HA or Myc antibody. The immunoprecipitates were detected by Western blot ( WB ) with anti-Myc and anti-HA antibodies, respectively. B , co-immunoprecipitation of endogenous PHF1 and p53 proteins in U2OS cells. The endogenous p53 was immunoprecipitated by p53 (FL-393) antibody with rabbit IgG as a negative control. The immunoprecipitates were detected by Western blot with anti-PHF1 and p53 antibodies, respectively. C , H1299 cells were transfected with Myc-p53 and/or ( .) GFP-PHF1. Cells were fixed 48 h after transfection and subjected to immunofluorescence analysis. D , bacterially expressed GST fusion PHF1 protein bound to glutathione-Sepharose beads and incubated with His-tagged fusion p53 protein. Bound His-p53 was detected by Western blot with anti-His antibody. M.W. , molecular weight.

Techniques Used: In Vivo, In Vitro, Transfection, Immunoprecipitation, Western Blot, Negative Control, Immunofluorescence, Incubation, Molecular Weight

Delineation of the domains mediating the mutual interactions between PHF1 and p53. A , schematic presentation of p53 domains ( left panel , top ) and deletion mutants ( left panel , bottom ) and the results of GST pulldown experiments ( right panel ). Bacterially expressed GST fusion proteins of wild-type p53 and the deletion mutants were bound to glutathione-Sepharose beads, respectively, and incubated with lysates of H1299 cells transfected with a Myc-PHF1 expression construct. Bound Myc-PHF1 was detected by Western blot with anti-HA antibody, and various GST-p53 proteins were detected by ponceau S staining. B and C , schematic model of PHF1 domains ( left panel , top ) and deletion mutants ( left panel , bottom ) and the results of GST pulldown experiments ( right panel ). Bacterially expressed GST fusion proteins of wild-type PHF1 and the deletion mutants were bound to glutathione-Sepharose beads, respectively, and incubated with lysates of H1299 cells transfected with a Myc-p53 expression construct. Bound Myc-p53 was detected by Western blot with Do-1 antibody, and GST-PHF1 deletion mutant proteins were detected by ponceau S staining. M.W. , molecular weight.
Figure Legend Snippet: Delineation of the domains mediating the mutual interactions between PHF1 and p53. A , schematic presentation of p53 domains ( left panel , top ) and deletion mutants ( left panel , bottom ) and the results of GST pulldown experiments ( right panel ). Bacterially expressed GST fusion proteins of wild-type p53 and the deletion mutants were bound to glutathione-Sepharose beads, respectively, and incubated with lysates of H1299 cells transfected with a Myc-PHF1 expression construct. Bound Myc-PHF1 was detected by Western blot with anti-HA antibody, and various GST-p53 proteins were detected by ponceau S staining. B and C , schematic model of PHF1 domains ( left panel , top ) and deletion mutants ( left panel , bottom ) and the results of GST pulldown experiments ( right panel ). Bacterially expressed GST fusion proteins of wild-type PHF1 and the deletion mutants were bound to glutathione-Sepharose beads, respectively, and incubated with lysates of H1299 cells transfected with a Myc-p53 expression construct. Bound Myc-p53 was detected by Western blot with Do-1 antibody, and GST-PHF1 deletion mutant proteins were detected by ponceau S staining. M.W. , molecular weight.

Techniques Used: Incubation, Transfection, Expressing, Construct, Western Blot, Staining, Mutagenesis, Molecular Weight

74) Product Images from "The Four Trypanosomatid eIF4E Homologues Fall into Two Separate Groups, with Distinct Features in Primary Sequence and Biological Properties"

Article Title: The Four Trypanosomatid eIF4E Homologues Fall into Two Separate Groups, with Distinct Features in Primary Sequence and Biological Properties

Journal: Molecular and biochemical parasitology

doi: 10.1016/j.molbiopara.2010.11.011

Analysis of the interactions between Tb EIF4E3/ Tb EIF4E4 and Tb EIF4G3/ Tb EIF4G4. (A) Pull-down assay using GST-tagged Tb EIF4G3 and 4 and 35 S-labelled Tb EIF4E1 through Tb EIF4E4. As controls GST on its own or only the glutathione sepharose beads were tested for their ability to bind the labeled protein. Upper panel: Coomassie Blue stained gel showing total translation extract (Input) as well as recombinant GST or GST- Tb EIF4G3 and 4. Bottom panels: autoradiographies showing strong and specific binding between GST- Tb EIF4G3 and labeled Tb EIF4E4 and between GST- Tb EIF4G4 and labeled Tb EIF4E3. (B) Immunoprecipitation (IP) assay confirming interactions between Tb EIF4E3 and Tb EIF4G4 and between Tb EIF4E3 and Tb EIF4G3 in vivo . T. brucei cytoplasmic extracts were immunoprecipitated with the anti- Tb EIF4E3 affinity purified antibodies (2 μg) or 5 μl of the pre-immune total serum as control (Control). Precipitated immunocomplexes were then used in Western blot assays with the Tb EIF4E3, Tb EIF4G3 or Tb EIF4G4 antisera. In the same blots, an aliquot of the non-bound supernatant (Supernatant) was compared with an equivalent aliquot of the cytoplasmic extract used in the IP reaction and blot (Input). Equivalent aliquot means that if 20% of an IP was blotted (the real % values varied according to the protein assayed), then 20% of the same volume of the original cytoplasmic extract used for the IP, or its supernatant, was blotted as well. (C) Assay confirming the interaction between Tb EIF4E4 and Tb EIF4G3 in vivo . Immunoprecipitation was carried out as described in B with anti- Tb EIF4E4 antibodies followed by Western-blots with the Tb EIF4E4 and Tb EIF4G3 antisera. (D) IP of HA-tagged eIF4E homologues. Cytoplasmic extracts from transfected T. brucei cells expressing HA-tagged Tb EIF4E1, 3 and 4 (left, middle and right panels, respectively) were incubated with anti-HA beads and the precipitated samples, as well as equivalent aliquots of the non-bound supernatant and the input extract, were assayed for the presence of the HA-tagged proteins, Tb EIF4G3 and 4 and Tb EIF4AI. (E) To highlight the results from the IP lanes shown in D, these were ran on another gel and blotted again with the Tb EIF4G3 and 4 and Tb EIF4AI antisera, confirming the potential for both Tb EIF4E3 and 4 to reconstitute functional eIF4F complexes.
Figure Legend Snippet: Analysis of the interactions between Tb EIF4E3/ Tb EIF4E4 and Tb EIF4G3/ Tb EIF4G4. (A) Pull-down assay using GST-tagged Tb EIF4G3 and 4 and 35 S-labelled Tb EIF4E1 through Tb EIF4E4. As controls GST on its own or only the glutathione sepharose beads were tested for their ability to bind the labeled protein. Upper panel: Coomassie Blue stained gel showing total translation extract (Input) as well as recombinant GST or GST- Tb EIF4G3 and 4. Bottom panels: autoradiographies showing strong and specific binding between GST- Tb EIF4G3 and labeled Tb EIF4E4 and between GST- Tb EIF4G4 and labeled Tb EIF4E3. (B) Immunoprecipitation (IP) assay confirming interactions between Tb EIF4E3 and Tb EIF4G4 and between Tb EIF4E3 and Tb EIF4G3 in vivo . T. brucei cytoplasmic extracts were immunoprecipitated with the anti- Tb EIF4E3 affinity purified antibodies (2 μg) or 5 μl of the pre-immune total serum as control (Control). Precipitated immunocomplexes were then used in Western blot assays with the Tb EIF4E3, Tb EIF4G3 or Tb EIF4G4 antisera. In the same blots, an aliquot of the non-bound supernatant (Supernatant) was compared with an equivalent aliquot of the cytoplasmic extract used in the IP reaction and blot (Input). Equivalent aliquot means that if 20% of an IP was blotted (the real % values varied according to the protein assayed), then 20% of the same volume of the original cytoplasmic extract used for the IP, or its supernatant, was blotted as well. (C) Assay confirming the interaction between Tb EIF4E4 and Tb EIF4G3 in vivo . Immunoprecipitation was carried out as described in B with anti- Tb EIF4E4 antibodies followed by Western-blots with the Tb EIF4E4 and Tb EIF4G3 antisera. (D) IP of HA-tagged eIF4E homologues. Cytoplasmic extracts from transfected T. brucei cells expressing HA-tagged Tb EIF4E1, 3 and 4 (left, middle and right panels, respectively) were incubated with anti-HA beads and the precipitated samples, as well as equivalent aliquots of the non-bound supernatant and the input extract, were assayed for the presence of the HA-tagged proteins, Tb EIF4G3 and 4 and Tb EIF4AI. (E) To highlight the results from the IP lanes shown in D, these were ran on another gel and blotted again with the Tb EIF4G3 and 4 and Tb EIF4AI antisera, confirming the potential for both Tb EIF4E3 and 4 to reconstitute functional eIF4F complexes.

Techniques Used: Pull Down Assay, Labeling, Staining, Recombinant, Binding Assay, Immunoprecipitation, In Vivo, Affinity Purification, Western Blot, Transfection, Expressing, Incubation, Functional Assay

Cap binding assays. Autoradiography of the cap binding chromatography performed with the four 35 S-labelled T. brucei eIF4E homologues, the Leishmania Lm EIF4E4 and the Xenopus leavis eIF4E homologue as positive control. Aliquots of the various washes were ran on SDS-PAGE and compared with samples from the original translation reaction (Input) as well as the non-bound fraction (Flow-through) and any protein remaining bound to the beads (Beads). The arrows indicate the proteins eluted by the cap analogue. The assay shows the affinity of Tb EIF4E1, 2 and 4, as well as Lm EIF4E4, for the 7-methyl-GTP Sepharose resin.
Figure Legend Snippet: Cap binding assays. Autoradiography of the cap binding chromatography performed with the four 35 S-labelled T. brucei eIF4E homologues, the Leishmania Lm EIF4E4 and the Xenopus leavis eIF4E homologue as positive control. Aliquots of the various washes were ran on SDS-PAGE and compared with samples from the original translation reaction (Input) as well as the non-bound fraction (Flow-through) and any protein remaining bound to the beads (Beads). The arrows indicate the proteins eluted by the cap analogue. The assay shows the affinity of Tb EIF4E1, 2 and 4, as well as Lm EIF4E4, for the 7-methyl-GTP Sepharose resin.

Techniques Used: Binding Assay, Autoradiography, Chromatography, Positive Control, SDS Page, Flow Cytometry

75) Product Images from "The C-terminal region of the motor protein MCAK controls its structure and activity through a conformational switch"

Article Title: The C-terminal region of the motor protein MCAK controls its structure and activity through a conformational switch

Journal: eLife

doi: 10.7554/eLife.06421

Sequence requirement for the formation of a motor-CT tail complex. ( A ) Sequence alignment of the conserved CT domain of MCAK for various species alongside the Drosophila kinesin-13 Klp10A and human Kif2a. The conserved residues are highlighted in red. The three amino acids that are critical for binding to the motor domain are marked with a green star. ( B ) Sequence alignment of the C terminus of human Kif2a, Kif2b, and MCAK/Kif2c. Amino acid numbering is relative to the Kif2a sequence. The MCAK CT domain binding to the motor domain is boxed in green. The sequences were aligned using the program T-coffee (EBI) and formatted with ESPRIPT ( Gouet et al., 1999 ). ( C ) Coomassie-stained gel showing a resin-based binding assay using glutathione agarose beads for purified His-M, binding to the GST-CT and GST-CT point mutants. ( D ) Size-exclusion chromatography elution profile of the motor domain alone (red dashes), motor incubated with the CT, CT S715E , CT E711A, E712A domains (cyan, green dashes, and purple, respectively). Bottom, coomassie-stained gel showing the size-exclusion chromatography elution of the motor incubated with the CT S715E and CT E711A, E712A domains (green and purple, respectively). DOI: http://dx.doi.org/10.7554/eLife.06421.008
Figure Legend Snippet: Sequence requirement for the formation of a motor-CT tail complex. ( A ) Sequence alignment of the conserved CT domain of MCAK for various species alongside the Drosophila kinesin-13 Klp10A and human Kif2a. The conserved residues are highlighted in red. The three amino acids that are critical for binding to the motor domain are marked with a green star. ( B ) Sequence alignment of the C terminus of human Kif2a, Kif2b, and MCAK/Kif2c. Amino acid numbering is relative to the Kif2a sequence. The MCAK CT domain binding to the motor domain is boxed in green. The sequences were aligned using the program T-coffee (EBI) and formatted with ESPRIPT ( Gouet et al., 1999 ). ( C ) Coomassie-stained gel showing a resin-based binding assay using glutathione agarose beads for purified His-M, binding to the GST-CT and GST-CT point mutants. ( D ) Size-exclusion chromatography elution profile of the motor domain alone (red dashes), motor incubated with the CT, CT S715E , CT E711A, E712A domains (cyan, green dashes, and purple, respectively). Bottom, coomassie-stained gel showing the size-exclusion chromatography elution of the motor incubated with the CT S715E and CT E711A, E712A domains (green and purple, respectively). DOI: http://dx.doi.org/10.7554/eLife.06421.008

Techniques Used: Sequencing, Binding Assay, Staining, Purification, Size-exclusion Chromatography, Incubation

The C terminus of MCAK binds to the motor domain. ( A ) Top: schematic diagram showing the different functional domains of full-length MCAK. Bottom: table representing the constructs used and given names. ( B ) Coomassie-stained gel showing a resin-based binding assay for purified His-M, His-NM, and CT domains to either glutathione agarose beads containing GST (as a control) or the GST-CT domain. The star represents residual GST. ( C ) Top, gel filtration elution profile of MCAK motor alone (M, red) and MCAK motor bound to the CT domain (M + CT, cyan). Bottom, coomassie-stained gel showing the size-exclusion chromatography profile of M and M + CT. DOI: http://dx.doi.org/10.7554/eLife.06421.003
Figure Legend Snippet: The C terminus of MCAK binds to the motor domain. ( A ) Top: schematic diagram showing the different functional domains of full-length MCAK. Bottom: table representing the constructs used and given names. ( B ) Coomassie-stained gel showing a resin-based binding assay for purified His-M, His-NM, and CT domains to either glutathione agarose beads containing GST (as a control) or the GST-CT domain. The star represents residual GST. ( C ) Top, gel filtration elution profile of MCAK motor alone (M, red) and MCAK motor bound to the CT domain (M + CT, cyan). Bottom, coomassie-stained gel showing the size-exclusion chromatography profile of M and M + CT. DOI: http://dx.doi.org/10.7554/eLife.06421.003

Techniques Used: Functional Assay, Construct, Staining, Binding Assay, Purification, Filtration, Size-exclusion Chromatography

Related Articles

Transduction:

Article Title: Identification of Yin-Yang Regulators and a Phosphorylation Consensus for Male Germ Cell-Associated Kinase (MAK)-Related Kinase ▿
Article Snippet: The rest of the cell lysate was incubated with glutathione-Sepharose beads (Amersham Biosciences) for 2 h at 4°C to absorb GST fusion proteins. .. The lysate and bead eluates were subjected to Western blotting against the following antibodies: 0.2 μg/ml anti-GST (Santa Cruz B-14), 0.1 μg/ml anti-Flag M2 (Sigma), 0.1 μg/ml anti-HA (Sigma) and 2.0 μg/ml mouse anti-protein phosphatase 5 (BD Transduction Laboratories).

Clone Assay:

Article Title: The V Protein of Tioman Virus Is Incapable of Blocking Type I Interferon Signaling in Human Cells
Article Snippet: Co-affinity Purification Experiments and Western Blot Analysis To perform co-affinity purification experiments, cloned ORFs were transferred from pDONR207 to pDEST27 expression vector (Invitrogen) to achieve GST fusion, and to pCI-neo-3xFLAG vector for 3xFLAG fusion. .. Cell lysates were incubated on ice for 20 min, then clarified by centrifugation at 14,000×g for 10 min. For pull-down analysis, 400 µg of protein extracts were incubated for 1 h at 4°C with 25 µl of glutathione-sepharose beads (Amersham Biosciences) to purify GST-tagged proteins.

Article Title: Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5 *
Article Snippet: Approximately 20 independent clones were screened to test NHE51D4 expression by Western blot and immunofluorescence microscopy, and several independent clones expressing moderate levels of NHE5 were analyzed. .. GST fusion proteins were purified by incubation with reduced form glutathione-Sepharose beads (Amersham Biosciences) at 4 °C.

Article Title: RARα2 and PML-RAR similarities in the control of basal and retinoic acid induced myeloid maturation of acute myeloid leukemia cells
Article Snippet: GST-RARα2 was obtained from E. coli cells transformed with an appropriate RARα2 cDNA construct cloned in the EcoRI-Not1 sites of pGEX4T2 . .. The recombinant proteins conjugated to Glutathione-sepharose beads (Amersham) were incubated with extracts of COS-7 cells transfected with pHA-RARα2 , pHA-SNAIL , pcDNA3-RARα1 , pSG5-PML-RAR or pcDNA3 containing the HA-tag (pHA ) and pcDNA3 plasmids, for 4 hours.

Centrifugation:

Article Title: The V Protein of Tioman Virus Is Incapable of Blocking Type I Interferon Signaling in Human Cells
Article Snippet: .. Cell lysates were incubated on ice for 20 min, then clarified by centrifugation at 14,000×g for 10 min. For pull-down analysis, 400 µg of protein extracts were incubated for 1 h at 4°C with 25 µl of glutathione-sepharose beads (Amersham Biosciences) to purify GST-tagged proteins. .. Beads were then washed 3 times in ice-cold lysis buffer and proteins were recovered by boiling in denaturing loading buffer (Invitrogen).

Article Title: Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5 *
Article Snippet: Cell lysates were then incubated for 30 min on ice and then sonicated four times for 30 s. After sonication, cell debris was cleared by centrifugation for 10 min at 16,000 × g at 4 °C. .. GST fusion proteins were purified by incubation with reduced form glutathione-Sepharose beads (Amersham Biosciences) at 4 °C.

Article Title: The Sec34/Sec35p complex, a Ypt1p effector required for retrograde intra-Golgi trafficking, interacts with Golgi SNAREs and COPI vesicle coat proteins
Article Snippet: .. This membrane extract (1 ml) was cleared by centrifugation at 20,000 g for 10 min at 4°C and mixed with 50 μl prewashed glutathione–Sepharose beads (Amersham Pharmacia Biotech). .. After a 30-min incubation (4°C, rotation), the beads were washed four times with 20 vol of washing buffer (20 mM Hepes, pH 7.4, 0.1% CHAPS, 0.15 mM NaCl).

Article Title: E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue
Article Snippet: The lysates were cleared by centrifugation, and the supernatants, containing the His-tagged proteins, were incubated with Ni-NTA agarose (Qiagen, Hilden, Germany) for 1 h at 4°C. .. The GST-tagged proteins were then purified from the cleared lysates by affinity purification using Glutathione Sepharose beads (GE Healthcare Life Sciences).

Article Title: Identification of Yin-Yang Regulators and a Phosphorylation Consensus for Male Germ Cell-Associated Kinase (MAK)-Related Kinase ▿
Article Snippet: The cell lysate was cleared by centrifugation. .. The rest of the cell lysate was incubated with glutathione-Sepharose beads (Amersham Biosciences) for 2 h at 4°C to absorb GST fusion proteins.

Article Title:
Article Snippet: Cells expressing GST-tagged nuclear transport factors, GST-tagged NLSs, and GST-AID were harvested by centrifugation and lysed by sonication in buffer A, B, or C respectively ( supplemental Table S2 ). .. Cleared lysates were incubated with glutathione-Sepharose beads (Amersham Biosciences) to purify GST-tagged proteins.

Luciferase:

Article Title: Two Distinct Calmodulin Binding Sites in the Third Intracellular Loop and Carboxyl Tail of Angiotensin II (AT1A) Receptor
Article Snippet: Materials GST expression vector pGEX-4T-1 and Glutathione-Sepharose 4B beads were purchased from Amersham Biosciences (Piscataway, NJ). .. The yellow fluorescent protein fusion protein expression vector eYFP-N1 and the Renilla luciferase (RLuc) protein fusion protein expression vector were purchased from Clontech (Mountain View, CA).

Filtration:

Article Title:
Article Snippet: Cleared lysates were incubated with glutathione-Sepharose beads (Amersham Biosciences) to purify GST-tagged proteins. .. Peak fractions were further purified by anion exchange at pH 8.0 on a Q column (GE Healthcare) and subsequent gel filtration on Superdex 75 (GE Healthcare).

Stable Transfection:

Article Title: Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5 *
Article Snippet: NHE51D4 was transfected to PC12 cells using the conventional calcium phosphate method , and cells stably expressing NHE51D4 were selected in selection media containing G418 (200 μg/ml). .. GST fusion proteins were purified by incubation with reduced form glutathione-Sepharose beads (Amersham Biosciences) at 4 °C.

Synthesized:

Article Title: Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap
Article Snippet: .. Purified GST, GST.pVIII, or GST.DDX3 fusion protein (15 μg each) was incubated individually with 20 μl of glutathione sepharose beads (GE Health Care) plus 10 μl of in vitro synthesized indicated proteins at +4°C on a nutator. .. After overnight incubation, the beads were washed three times, 10 min each with 0.1 M PBS.

Construct:

Article Title: RARα2 and PML-RAR similarities in the control of basal and retinoic acid induced myeloid maturation of acute myeloid leukemia cells
Article Snippet: GST-RARα2 was obtained from E. coli cells transformed with an appropriate RARα2 cDNA construct cloned in the EcoRI-Not1 sites of pGEX4T2 . .. The recombinant proteins conjugated to Glutathione-sepharose beads (Amersham) were incubated with extracts of COS-7 cells transfected with pHA-RARα2 , pHA-SNAIL , pcDNA3-RARα1 , pSG5-PML-RAR or pcDNA3 containing the HA-tag (pHA ) and pcDNA3 plasmids, for 4 hours.

Article Title: ORF73 of Herpesvirus Saimiri, a Viral Homolog of Kaposi's Sarcoma-Associated Herpesvirus, Modulates the Two Cellular Tumor Suppressor Proteins p53 and pRb
Article Snippet: HVS ORF73 constructs were in vitro transcribed and translated with 5 μg of plasmid template and either 35 S-labeled methionine or 14 C-labeled leucine (PerkinElmer). .. For GST pulldown assays, GST, GST-p53, or GST-pRb protein was expressed in Escherichia coli DH5α cells and purified by incubation of sonicated cell lysate with glutathione-Sepharose beads (Amersham Pharmacia, Inc., Uppsala, Sweden) for 6 h at 4°C under constant rotation.

Electrophoresis:

Article Title: The Sec34/Sec35p complex, a Ypt1p effector required for retrograde intra-Golgi trafficking, interacts with Golgi SNAREs and COPI vesicle coat proteins
Article Snippet: Paragraph title: Purification of GST-tagged proteins from the yeast, electrophoresis, and immunoblot analysis ... This membrane extract (1 ml) was cleared by centrifugation at 20,000 g for 10 min at 4°C and mixed with 50 μl prewashed glutathione–Sepharose beads (Amersham Pharmacia Biotech).

Incubation:

Article Title: The V Protein of Tioman Virus Is Incapable of Blocking Type I Interferon Signaling in Human Cells
Article Snippet: .. Cell lysates were incubated on ice for 20 min, then clarified by centrifugation at 14,000×g for 10 min. For pull-down analysis, 400 µg of protein extracts were incubated for 1 h at 4°C with 25 µl of glutathione-sepharose beads (Amersham Biosciences) to purify GST-tagged proteins. .. Beads were then washed 3 times in ice-cold lysis buffer and proteins were recovered by boiling in denaturing loading buffer (Invitrogen).

Article Title: Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5 *
Article Snippet: .. GST fusion proteins were purified by incubation with reduced form glutathione-Sepharose beads (Amersham Biosciences) at 4 °C. .. GST Pulldown —A 35 S-labeled NHE5 C-terminal domain (Gly491 -Leu896 ) was produced by in vitro transcription-translation using the T n T-coupled reticulocyte lysate system (Promega, Madison, WI) according to the manufacturer's instructions.

Article Title: The Sec34/Sec35p complex, a Ypt1p effector required for retrograde intra-Golgi trafficking, interacts with Golgi SNAREs and COPI vesicle coat proteins
Article Snippet: The pellet was suspended in extraction buffer (20 mM Hepes, pH 7.4, 1% CHAPS, 0.15 M NaCl) and incubated on ice for 20 min. .. This membrane extract (1 ml) was cleared by centrifugation at 20,000 g for 10 min at 4°C and mixed with 50 μl prewashed glutathione–Sepharose beads (Amersham Pharmacia Biotech).

Article Title: E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue
Article Snippet: The lysates were cleared by centrifugation, and the supernatants, containing the His-tagged proteins, were incubated with Ni-NTA agarose (Qiagen, Hilden, Germany) for 1 h at 4°C. .. The GST-tagged proteins were then purified from the cleared lysates by affinity purification using Glutathione Sepharose beads (GE Healthcare Life Sciences).

Article Title: RARα2 and PML-RAR similarities in the control of basal and retinoic acid induced myeloid maturation of acute myeloid leukemia cells
Article Snippet: .. The recombinant proteins conjugated to Glutathione-sepharose beads (Amersham) were incubated with extracts of COS-7 cells transfected with pHA-RARα2 , pHA-SNAIL , pcDNA3-RARα1 , pSG5-PML-RAR or pcDNA3 containing the HA-tag (pHA ) and pcDNA3 plasmids, for 4 hours. .. Pulled-down proteins were subjected to WB analysis using anti-HA, anti-RARα or anti-GST antibodies.

Article Title: Complexes between the LKB1 tumor suppressor, STRAD?/? and MO25?/? are upstream kinases in the AMP-activated protein kinase cascade
Article Snippet: The E. coli lysate expressing GST-AMPKα1 was adsorbed onto glutathione-Sepharose beads (Amersham-Pharmacia) such that the final concentration of kinase after maximal activation using MgATP and AMPKK in the assay below was 1 unit in the standard kinase assay per 5 μl of beads. .. For the kinase kinase assay, the AMPKK preparation was incubated with 10 μl of a 50% slurry of the glutathione-Sepharose beads with bound GST-AMPKα1, plus 200 μM AMP, 200 μM ATP, 5 mM MgCl2 in assay buffer in a final volume of 25 μl.

Article Title: Identification of Yin-Yang Regulators and a Phosphorylation Consensus for Male Germ Cell-Associated Kinase (MAK)-Related Kinase ▿
Article Snippet: .. The rest of the cell lysate was incubated with glutathione-Sepharose beads (Amersham Biosciences) for 2 h at 4°C to absorb GST fusion proteins. .. The beads were washed extensively with lysis buffer followed by phosphate-buffered saline buffer.

Article Title:
Article Snippet: .. Cleared lysates were incubated with glutathione-Sepharose beads (Amersham Biosciences) to purify GST-tagged proteins. .. E. coli -expressing recombinant His-CTNNBL1 and His-CTNNBL1(Δ1–76) were lysed by sonication in buffer D (Table S2 ), and the recombinant proteins were purified by binding onto a nickel-nitrilotriacetic acid column (GE Healthcare) followed by elution with a 0–500 mm imidazole gradient.

Article Title: ORF73 of Herpesvirus Saimiri, a Viral Homolog of Kaposi's Sarcoma-Associated Herpesvirus, Modulates the Two Cellular Tumor Suppressor Proteins p53 and pRb
Article Snippet: .. For GST pulldown assays, GST, GST-p53, or GST-pRb protein was expressed in Escherichia coli DH5α cells and purified by incubation of sonicated cell lysate with glutathione-Sepharose beads (Amersham Pharmacia, Inc., Uppsala, Sweden) for 6 h at 4°C under constant rotation. ..

Article Title: E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue
Article Snippet: .. For the in vitro pVHL ubiquitination assay, reaction mixtures were incubated 0.5 μg of His-E1, 0.2 μg of UCP, 2 μg of pVHL and 1.25 μg Flag-ubiquitin in reaction buffer along with the ATP regeneration system at 37°C for 1 h. To precipitate the ubiquitinated pVHL, the total reaction mixtures were pulled down with Glutathione Sepharose beads or Ni-NTA agarose on a rotary shaker at 4°C for 3 h. The beads were then washed three times under reducing (1% NP40 in NET gel buffer) or non-reducing (4 M urea and 1% NP40 in NET gel buffer) conditions and resuspended in 2X SDS sample buffer under denaturing conditions. .. For the in vivo ubiquitination assay, cells were co-transfected with indicated plasmids and treated with 10 μM MG132 for 12 h before harvesting.

Article Title: Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap
Article Snippet: .. Purified GST, GST.pVIII, or GST.DDX3 fusion protein (15 μg each) was incubated individually with 20 μl of glutathione sepharose beads (GE Health Care) plus 10 μl of in vitro synthesized indicated proteins at +4°C on a nutator. .. After overnight incubation, the beads were washed three times, 10 min each with 0.1 M PBS.

Expressing:

Article Title: The V Protein of Tioman Virus Is Incapable of Blocking Type I Interferon Signaling in Human Cells
Article Snippet: Co-affinity Purification Experiments and Western Blot Analysis To perform co-affinity purification experiments, cloned ORFs were transferred from pDONR207 to pDEST27 expression vector (Invitrogen) to achieve GST fusion, and to pCI-neo-3xFLAG vector for 3xFLAG fusion. .. Cell lysates were incubated on ice for 20 min, then clarified by centrifugation at 14,000×g for 10 min. For pull-down analysis, 400 µg of protein extracts were incubated for 1 h at 4°C with 25 µl of glutathione-sepharose beads (Amersham Biosciences) to purify GST-tagged proteins.

Article Title: Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5 *
Article Snippet: Protein expression was induced by incubating transformed BL21 Escherichia coli cells with 0.2 m m isopropyl 1-thio-β- d -galactopyranoside at 37 °C for 3 h. E. coli cells were collected by centrifugation and resuspended in lysis buffer containing 1% Triton X-100 and protease inhibitor mixture (Roche Diagnostics, Laval, Canada) in PBS. .. GST fusion proteins were purified by incubation with reduced form glutathione-Sepharose beads (Amersham Biosciences) at 4 °C.

Article Title: The Sec34/Sec35p complex, a Ypt1p effector required for retrograde intra-Golgi trafficking, interacts with Golgi SNAREs and COPI vesicle coat proteins
Article Snippet: 5 ml of an overnight culture grown in selective medium containing 1% glucose was used to inoculate 100 ml fresh selective medium containing 2% galactose to induce the expression of the GST fusion proteins. .. This membrane extract (1 ml) was cleared by centrifugation at 20,000 g for 10 min at 4°C and mixed with 50 μl prewashed glutathione–Sepharose beads (Amersham Pharmacia Biotech).

Article Title: Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap
Article Snippet: Paragraph title: Recombinant Protein Expression and Protein Purification ... Glutathione S-transferase (GST), GST.pVIII, and GST.DDX3 fusion proteins were purified using Glutathione sepharose beads (GE Healthcare) as per the instructions of the manufacturers.

Article Title: Herpes Simplex Virus ICP27 Protein Directly Interacts with the Nuclear Pore Complex through Nup62, Inhibiting Host Nucleocytoplasmic Transport Pathways *
Article Snippet: Paragraph title: Protein Expression in Escherichia coli ... GST-ICP27 , GST-Nup62, GST-RaeI, and GST alone recombinant proteins were expressed in E. coli BL21 cells and purified on glutathione-Sepharose beads (GE Healthcare) as described ( ).

Article Title: Complexes between the LKB1 tumor suppressor, STRAD?/? and MO25?/? are upstream kinases in the AMP-activated protein kinase cascade
Article Snippet: .. The E. coli lysate expressing GST-AMPKα1 was adsorbed onto glutathione-Sepharose beads (Amersham-Pharmacia) such that the final concentration of kinase after maximal activation using MgATP and AMPKK in the assay below was 1 unit in the standard kinase assay per 5 μl of beads. ..

Article Title: Two Distinct Calmodulin Binding Sites in the Third Intracellular Loop and Carboxyl Tail of Angiotensin II (AT1A) Receptor
Article Snippet: .. Materials GST expression vector pGEX-4T-1 and Glutathione-Sepharose 4B beads were purchased from Amersham Biosciences (Piscataway, NJ). .. The E. coli BL21 gold strain was purchased from Stratagene (La Jolla, CA).

Article Title:
Article Snippet: Cells expressing GST-tagged nuclear transport factors, GST-tagged NLSs, and GST-AID were harvested by centrifugation and lysed by sonication in buffer A, B, or C respectively ( supplemental Table S2 ). .. Cleared lysates were incubated with glutathione-Sepharose beads (Amersham Biosciences) to purify GST-tagged proteins.

Bradford Assay:

Article Title: Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap
Article Snippet: Glutathione S-transferase (GST), GST.pVIII, and GST.DDX3 fusion proteins were purified using Glutathione sepharose beads (GE Healthcare) as per the instructions of the manufacturers. .. The concentrations of the proteins were measured by Bradford assay (Bio Rad) using Ultrospec® 3000 spectrophotometer (Pharmacia Biotech).

Western Blot:

Article Title: The V Protein of Tioman Virus Is Incapable of Blocking Type I Interferon Signaling in Human Cells
Article Snippet: Paragraph title: Co-affinity Purification Experiments and Western Blot Analysis ... Cell lysates were incubated on ice for 20 min, then clarified by centrifugation at 14,000×g for 10 min. For pull-down analysis, 400 µg of protein extracts were incubated for 1 h at 4°C with 25 µl of glutathione-sepharose beads (Amersham Biosciences) to purify GST-tagged proteins.

Article Title: Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5 *
Article Snippet: Approximately 20 independent clones were screened to test NHE51D4 expression by Western blot and immunofluorescence microscopy, and several independent clones expressing moderate levels of NHE5 were analyzed. .. GST fusion proteins were purified by incubation with reduced form glutathione-Sepharose beads (Amersham Biosciences) at 4 °C.

Article Title: RARα2 and PML-RAR similarities in the control of basal and retinoic acid induced myeloid maturation of acute myeloid leukemia cells
Article Snippet: The recombinant proteins conjugated to Glutathione-sepharose beads (Amersham) were incubated with extracts of COS-7 cells transfected with pHA-RARα2 , pHA-SNAIL , pcDNA3-RARα1 , pSG5-PML-RAR or pcDNA3 containing the HA-tag (pHA ) and pcDNA3 plasmids, for 4 hours. .. Pulled-down proteins were subjected to WB analysis using anti-HA, anti-RARα or anti-GST antibodies.

Article Title: Identification of Yin-Yang Regulators and a Phosphorylation Consensus for Male Germ Cell-Associated Kinase (MAK)-Related Kinase ▿
Article Snippet: A portion of the cell lysate was saved for Western blotting to indicate protein signal input. .. The rest of the cell lysate was incubated with glutathione-Sepharose beads (Amersham Biosciences) for 2 h at 4°C to absorb GST fusion proteins.

Transformation Assay:

Article Title: Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5 *
Article Snippet: Protein expression was induced by incubating transformed BL21 Escherichia coli cells with 0.2 m m isopropyl 1-thio-β- d -galactopyranoside at 37 °C for 3 h. E. coli cells were collected by centrifugation and resuspended in lysis buffer containing 1% Triton X-100 and protease inhibitor mixture (Roche Diagnostics, Laval, Canada) in PBS. .. GST fusion proteins were purified by incubation with reduced form glutathione-Sepharose beads (Amersham Biosciences) at 4 °C.

Article Title: RARα2 and PML-RAR similarities in the control of basal and retinoic acid induced myeloid maturation of acute myeloid leukemia cells
Article Snippet: GST-RARα2 was obtained from E. coli cells transformed with an appropriate RARα2 cDNA construct cloned in the EcoRI-Not1 sites of pGEX4T2 . .. The recombinant proteins conjugated to Glutathione-sepharose beads (Amersham) were incubated with extracts of COS-7 cells transfected with pHA-RARα2 , pHA-SNAIL , pcDNA3-RARα1 , pSG5-PML-RAR or pcDNA3 containing the HA-tag (pHA ) and pcDNA3 plasmids, for 4 hours.

Kinase Assay:

Article Title: Complexes between the LKB1 tumor suppressor, STRAD?/? and MO25?/? are upstream kinases in the AMP-activated protein kinase cascade
Article Snippet: .. The E. coli lysate expressing GST-AMPKα1 was adsorbed onto glutathione-Sepharose beads (Amersham-Pharmacia) such that the final concentration of kinase after maximal activation using MgATP and AMPKK in the assay below was 1 unit in the standard kinase assay per 5 μl of beads. ..

Derivative Assay:

Article Title: RARα2 and PML-RAR similarities in the control of basal and retinoic acid induced myeloid maturation of acute myeloid leukemia cells
Article Snippet: For the pull-down experiments [ ], we used the described GST-RARα1 , GST-RARα2 and derived recombinant proteins. .. The recombinant proteins conjugated to Glutathione-sepharose beads (Amersham) were incubated with extracts of COS-7 cells transfected with pHA-RARα2 , pHA-SNAIL , pcDNA3-RARα1 , pSG5-PML-RAR or pcDNA3 containing the HA-tag (pHA ) and pcDNA3 plasmids, for 4 hours.

Transfection:

Article Title: The V Protein of Tioman Virus Is Incapable of Blocking Type I Interferon Signaling in Human Cells
Article Snippet: Two days after transfection, HEK-293T cells were washed in PBS, then resuspended in lysis buffer (0.5% Nonidet P-40, 20 mM Tris–HCl at pH 8, 120 mM NaCl and 1 mM EDTA) supplemented with Complete Protease Inhibitor Cocktail (Roche). .. Cell lysates were incubated on ice for 20 min, then clarified by centrifugation at 14,000×g for 10 min. For pull-down analysis, 400 µg of protein extracts were incubated for 1 h at 4°C with 25 µl of glutathione-sepharose beads (Amersham Biosciences) to purify GST-tagged proteins.

Article Title: Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5 *
Article Snippet: NHE51D4 was transfected to PC12 cells using the conventional calcium phosphate method , and cells stably expressing NHE51D4 were selected in selection media containing G418 (200 μg/ml). .. GST fusion proteins were purified by incubation with reduced form glutathione-Sepharose beads (Amersham Biosciences) at 4 °C.

Article Title: RARα2 and PML-RAR similarities in the control of basal and retinoic acid induced myeloid maturation of acute myeloid leukemia cells
Article Snippet: .. The recombinant proteins conjugated to Glutathione-sepharose beads (Amersham) were incubated with extracts of COS-7 cells transfected with pHA-RARα2 , pHA-SNAIL , pcDNA3-RARα1 , pSG5-PML-RAR or pcDNA3 containing the HA-tag (pHA ) and pcDNA3 plasmids, for 4 hours. .. Pulled-down proteins were subjected to WB analysis using anti-HA, anti-RARα or anti-GST antibodies.

Article Title: Identification of Yin-Yang Regulators and a Phosphorylation Consensus for Male Germ Cell-Associated Kinase (MAK)-Related Kinase ▿
Article Snippet: Forty-eight hours after transfection, cells were harvested in ice-cold phosphate-buffered saline and lysed in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 2 mM EGTA, and supplemented with complete protease inhibitors [Roche], 1 mM Na3 VO4 , 1 μM microcystin LR, and 5 mM β-glycerophosphate). .. The rest of the cell lysate was incubated with glutathione-Sepharose beads (Amersham Biosciences) for 2 h at 4°C to absorb GST fusion proteins.

Concentration Assay:

Article Title: E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue
Article Snippet: The purified proteins were then dissolved in SDS sample buffer and separated by SDS-PAGE to analyze their concentration and purity. .. The GST-tagged proteins were then purified from the cleared lysates by affinity purification using Glutathione Sepharose beads (GE Healthcare Life Sciences).

Article Title: Complexes between the LKB1 tumor suppressor, STRAD?/? and MO25?/? are upstream kinases in the AMP-activated protein kinase cascade
Article Snippet: .. The E. coli lysate expressing GST-AMPKα1 was adsorbed onto glutathione-Sepharose beads (Amersham-Pharmacia) such that the final concentration of kinase after maximal activation using MgATP and AMPKK in the assay below was 1 unit in the standard kinase assay per 5 μl of beads. ..

Protease Inhibitor:

Article Title: The V Protein of Tioman Virus Is Incapable of Blocking Type I Interferon Signaling in Human Cells
Article Snippet: Two days after transfection, HEK-293T cells were washed in PBS, then resuspended in lysis buffer (0.5% Nonidet P-40, 20 mM Tris–HCl at pH 8, 120 mM NaCl and 1 mM EDTA) supplemented with Complete Protease Inhibitor Cocktail (Roche). .. Cell lysates were incubated on ice for 20 min, then clarified by centrifugation at 14,000×g for 10 min. For pull-down analysis, 400 µg of protein extracts were incubated for 1 h at 4°C with 25 µl of glutathione-sepharose beads (Amersham Biosciences) to purify GST-tagged proteins.

Article Title: Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5 *
Article Snippet: Protein expression was induced by incubating transformed BL21 Escherichia coli cells with 0.2 m m isopropyl 1-thio-β- d -galactopyranoside at 37 °C for 3 h. E. coli cells were collected by centrifugation and resuspended in lysis buffer containing 1% Triton X-100 and protease inhibitor mixture (Roche Diagnostics, Laval, Canada) in PBS. .. GST fusion proteins were purified by incubation with reduced form glutathione-Sepharose beads (Amersham Biosciences) at 4 °C.

Polymerase Chain Reaction:

Article Title: Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5 *
Article Snippet: Expression and Purification of GST Fusion Proteins —For producing GST fusion proteins, PCR fragments corresponding to different regions of the SCAMP2 cytoplasmic domains were inserted into a pGEX-2T bacterial expression vector (Amersham Biosciences) in-frame with the N-terminal GST tag as described previously ( ). .. GST fusion proteins were purified by incubation with reduced form glutathione-Sepharose beads (Amersham Biosciences) at 4 °C.

Sonication:

Article Title: Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5 *
Article Snippet: Cell lysates were then incubated for 30 min on ice and then sonicated four times for 30 s. After sonication, cell debris was cleared by centrifugation for 10 min at 16,000 × g at 4 °C. .. GST fusion proteins were purified by incubation with reduced form glutathione-Sepharose beads (Amersham Biosciences) at 4 °C.

Article Title: E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue
Article Snippet: To purify GST-tagged recombinant proteins, cells were lysed in lysis buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2 HPO4 , 2 mM KH2 PO4 and 1 mM PMSF, pH 7.4) by sonication. .. The GST-tagged proteins were then purified from the cleared lysates by affinity purification using Glutathione Sepharose beads (GE Healthcare Life Sciences).

Article Title:
Article Snippet: Cells expressing GST-tagged nuclear transport factors, GST-tagged NLSs, and GST-AID were harvested by centrifugation and lysed by sonication in buffer A, B, or C respectively ( supplemental Table S2 ). .. Cleared lysates were incubated with glutathione-Sepharose beads (Amersham Biosciences) to purify GST-tagged proteins.

Article Title: ORF73 of Herpesvirus Saimiri, a Viral Homolog of Kaposi's Sarcoma-Associated Herpesvirus, Modulates the Two Cellular Tumor Suppressor Proteins p53 and pRb
Article Snippet: .. For GST pulldown assays, GST, GST-p53, or GST-pRb protein was expressed in Escherichia coli DH5α cells and purified by incubation of sonicated cell lysate with glutathione-Sepharose beads (Amersham Pharmacia, Inc., Uppsala, Sweden) for 6 h at 4°C under constant rotation. ..

Affinity Purification:

Article Title: E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue
Article Snippet: .. The GST-tagged proteins were then purified from the cleared lysates by affinity purification using Glutathione Sepharose beads (GE Healthcare Life Sciences). .. Cell culture and transient transfection HEK-293T and HeLa cells were cultured at 37°C in a humidified 5% CO2 atmosphere in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco, NY, USA) and 1X (v/v) antibiotics (Gibco).

Binding Assay:

Article Title: Identification of Yin-Yang Regulators and a Phosphorylation Consensus for Male Germ Cell-Associated Kinase (MAK)-Related Kinase ▿
Article Snippet: Paragraph title: GST pull-down binding assay. ... The rest of the cell lysate was incubated with glutathione-Sepharose beads (Amersham Biosciences) for 2 h at 4°C to absorb GST fusion proteins.

Article Title:
Article Snippet: Cleared lysates were incubated with glutathione-Sepharose beads (Amersham Biosciences) to purify GST-tagged proteins. .. E. coli -expressing recombinant His-CTNNBL1 and His-CTNNBL1(Δ1–76) were lysed by sonication in buffer D (Table S2 ), and the recombinant proteins were purified by binding onto a nickel-nitrilotriacetic acid column (GE Healthcare) followed by elution with a 0–500 mm imidazole gradient.

Article Title: ORF73 of Herpesvirus Saimiri, a Viral Homolog of Kaposi's Sarcoma-Associated Herpesvirus, Modulates the Two Cellular Tumor Suppressor Proteins p53 and pRb
Article Snippet: For GST pulldown assays, GST, GST-p53, or GST-pRb protein was expressed in Escherichia coli DH5α cells and purified by incubation of sonicated cell lysate with glutathione-Sepharose beads (Amersham Pharmacia, Inc., Uppsala, Sweden) for 6 h at 4°C under constant rotation. .. To control for nonspecific binding in the GST pulldown experiment, in vitro-translated product was first gently agitated with glutathione-Sepharose beads in NETN buffer (20 mM Tris, pH 8, 1 mM EDTA, 100 mM NaCl, 0.5% NP-40, 2 μg of aprotinin per ml, 1 μg of pepstatin A per ml, 1 mM phenylmethylsulfonyl fluoride, and 2 μg of leupeptin per ml) for 30 min at 4°C, followed by gentle agitation with GST-coupled glutathione-Sepharose beads for 1 h at 4°C.

Article Title: Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap
Article Snippet: Paragraph title: In vitro Binding Assay ... Purified GST, GST.pVIII, or GST.DDX3 fusion protein (15 μg each) was incubated individually with 20 μl of glutathione sepharose beads (GE Health Care) plus 10 μl of in vitro synthesized indicated proteins at +4°C on a nutator.

Immunofluorescence:

Article Title: Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5 *
Article Snippet: Approximately 20 independent clones were screened to test NHE51D4 expression by Western blot and immunofluorescence microscopy, and several independent clones expressing moderate levels of NHE5 were analyzed. .. GST fusion proteins were purified by incubation with reduced form glutathione-Sepharose beads (Amersham Biosciences) at 4 °C.

Nucleic Acid Electrophoresis:

Article Title: The V Protein of Tioman Virus Is Incapable of Blocking Type I Interferon Signaling in Human Cells
Article Snippet: Cell lysates were incubated on ice for 20 min, then clarified by centrifugation at 14,000×g for 10 min. For pull-down analysis, 400 µg of protein extracts were incubated for 1 h at 4°C with 25 µl of glutathione-sepharose beads (Amersham Biosciences) to purify GST-tagged proteins. .. Purified complexes and protein extracts were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on 4–12% NuPAGE Bis–Tris gels with MOPS running buffer (Invitrogen), and transferred to a nitrocellulose membrane.

Microscopy:

Article Title: Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5 *
Article Snippet: Approximately 20 independent clones were screened to test NHE51D4 expression by Western blot and immunofluorescence microscopy, and several independent clones expressing moderate levels of NHE5 were analyzed. .. GST fusion proteins were purified by incubation with reduced form glutathione-Sepharose beads (Amersham Biosciences) at 4 °C.

Purification:

Article Title: The V Protein of Tioman Virus Is Incapable of Blocking Type I Interferon Signaling in Human Cells
Article Snippet: Paragraph title: Co-affinity Purification Experiments and Western Blot Analysis ... Cell lysates were incubated on ice for 20 min, then clarified by centrifugation at 14,000×g for 10 min. For pull-down analysis, 400 µg of protein extracts were incubated for 1 h at 4°C with 25 µl of glutathione-sepharose beads (Amersham Biosciences) to purify GST-tagged proteins.

Article Title: Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5 *
Article Snippet: .. GST fusion proteins were purified by incubation with reduced form glutathione-Sepharose beads (Amersham Biosciences) at 4 °C. .. GST Pulldown —A 35 S-labeled NHE5 C-terminal domain (Gly491 -Leu896 ) was produced by in vitro transcription-translation using the T n T-coupled reticulocyte lysate system (Promega, Madison, WI) according to the manufacturer's instructions.

Article Title: The Sec34/Sec35p complex, a Ypt1p effector required for retrograde intra-Golgi trafficking, interacts with Golgi SNAREs and COPI vesicle coat proteins
Article Snippet: Paragraph title: Purification of GST-tagged proteins from the yeast, electrophoresis, and immunoblot analysis ... This membrane extract (1 ml) was cleared by centrifugation at 20,000 g for 10 min at 4°C and mixed with 50 μl prewashed glutathione–Sepharose beads (Amersham Pharmacia Biotech).

Article Title: E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue
Article Snippet: .. The GST-tagged proteins were then purified from the cleared lysates by affinity purification using Glutathione Sepharose beads (GE Healthcare Life Sciences). .. Cell culture and transient transfection HEK-293T and HeLa cells were cultured at 37°C in a humidified 5% CO2 atmosphere in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco, NY, USA) and 1X (v/v) antibiotics (Gibco).

Article Title: Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap
Article Snippet: .. Glutathione S-transferase (GST), GST.pVIII, and GST.DDX3 fusion proteins were purified using Glutathione sepharose beads (GE Healthcare) as per the instructions of the manufacturers. .. The purified proteins were dialyzed using Slide-A-Lyzer dialysis cassette (Thermo Scientific).

Article Title: Herpes Simplex Virus ICP27 Protein Directly Interacts with the Nuclear Pore Complex through Nup62, Inhibiting Host Nucleocytoplasmic Transport Pathways *
Article Snippet: .. GST-ICP27 , GST-Nup62, GST-RaeI, and GST alone recombinant proteins were expressed in E. coli BL21 cells and purified on glutathione-Sepharose beads (GE Healthcare) as described ( ). .. Histidine-tagged ICP27 from pET-ICP27 was expressed in BL21 DE3 and purified using Talon resin (Clontech) as described ( ).

Article Title:
Article Snippet: Recombinant Protein Purification Recombinant proteins were purified from Escherichia coli BL21(DE3) transformants that had been incubated at 16 °C in LB overnight following induction with 1 mm isopropyl 1-thio-β-d -galactopyranoside at an A 600 of 0.6. .. Cleared lysates were incubated with glutathione-Sepharose beads (Amersham Biosciences) to purify GST-tagged proteins.

Article Title: ORF73 of Herpesvirus Saimiri, a Viral Homolog of Kaposi's Sarcoma-Associated Herpesvirus, Modulates the Two Cellular Tumor Suppressor Proteins p53 and pRb
Article Snippet: .. For GST pulldown assays, GST, GST-p53, or GST-pRb protein was expressed in Escherichia coli DH5α cells and purified by incubation of sonicated cell lysate with glutathione-Sepharose beads (Amersham Pharmacia, Inc., Uppsala, Sweden) for 6 h at 4°C under constant rotation. ..

Article Title: Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap
Article Snippet: .. Purified GST, GST.pVIII, or GST.DDX3 fusion protein (15 μg each) was incubated individually with 20 μl of glutathione sepharose beads (GE Health Care) plus 10 μl of in vitro synthesized indicated proteins at +4°C on a nutator. .. After overnight incubation, the beads were washed three times, 10 min each with 0.1 M PBS.

Article Title: The third helix of the homeodomain of paired class homeodomain proteins acts as a recognition helix both for DNA and protein interactions
Article Snippet: .. Glutathione S -transferase (GST) pull-down assays GST fusion proteins purified from Escherichia coli LE392 or E.coli BL21-Star(DE3)pLysS (Invitrogen) extracts using glutathione–sepharose beads (Amersham Pharmacia Biotech) were used in pull-down assays as described previously ( ). .. Transient transfection assays NIH 3T3 fibroblasts (passage 123) (ATCC CRL 1658) were cultured in DMEM supplemented with 10% calf serum (HyClone, Logan, UT), penicillin (100 U/ml) and 100 μg/ml streptomycin (Life Technologies, Inc.).

Protein Purification:

Article Title: Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap
Article Snippet: Paragraph title: Recombinant Protein Expression and Protein Purification ... Glutathione S-transferase (GST), GST.pVIII, and GST.DDX3 fusion proteins were purified using Glutathione sepharose beads (GE Healthcare) as per the instructions of the manufacturers.

Article Title:
Article Snippet: Paragraph title: Recombinant Protein Purification ... Cleared lysates were incubated with glutathione-Sepharose beads (Amersham Biosciences) to purify GST-tagged proteins.

Protein Extraction:

Article Title: E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue
Article Snippet: Paragraph title: Recombinant protein extraction ... The GST-tagged proteins were then purified from the cleared lysates by affinity purification using Glutathione Sepharose beads (GE Healthcare Life Sciences).

Positron Emission Tomography:

Article Title: Herpes Simplex Virus ICP27 Protein Directly Interacts with the Nuclear Pore Complex through Nup62, Inhibiting Host Nucleocytoplasmic Transport Pathways *
Article Snippet: GST-ICP27 , GST-Nup62, GST-RaeI, and GST alone recombinant proteins were expressed in E. coli BL21 cells and purified on glutathione-Sepharose beads (GE Healthcare) as described ( ). .. Histidine-tagged ICP27 from pET-ICP27 was expressed in BL21 DE3 and purified using Talon resin (Clontech) as described ( ).

SDS Page:

Article Title: The V Protein of Tioman Virus Is Incapable of Blocking Type I Interferon Signaling in Human Cells
Article Snippet: Cell lysates were incubated on ice for 20 min, then clarified by centrifugation at 14,000×g for 10 min. For pull-down analysis, 400 µg of protein extracts were incubated for 1 h at 4°C with 25 µl of glutathione-sepharose beads (Amersham Biosciences) to purify GST-tagged proteins. .. Purified complexes and protein extracts were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on 4–12% NuPAGE Bis–Tris gels with MOPS running buffer (Invitrogen), and transferred to a nitrocellulose membrane.

Article Title: The Sec34/Sec35p complex, a Ypt1p effector required for retrograde intra-Golgi trafficking, interacts with Golgi SNAREs and COPI vesicle coat proteins
Article Snippet: This membrane extract (1 ml) was cleared by centrifugation at 20,000 g for 10 min at 4°C and mixed with 50 μl prewashed glutathione–Sepharose beads (Amersham Pharmacia Biotech). .. Samples for immunoblotting were separated by SDS-PAGE (12% acrylamide, except where noted), electrotransferred to nitrocellulose, and probed with the appropriate primary antibodies according to standard protocols ( ).

Article Title: E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue
Article Snippet: The purified proteins were then dissolved in SDS sample buffer and separated by SDS-PAGE to analyze their concentration and purity. .. The GST-tagged proteins were then purified from the cleared lysates by affinity purification using Glutathione Sepharose beads (GE Healthcare Life Sciences).

Plasmid Preparation:

Article Title: The V Protein of Tioman Virus Is Incapable of Blocking Type I Interferon Signaling in Human Cells
Article Snippet: Briefly, 5×105 HEK-293T cells were dispensed in each well of a 6-well plate, and transfected 24 h later with 500 ng of each pDEST27 plasmid encoding viral ORFs and 300 ng of pCI-neo-3xFLAG vector containing 3xFLAG-tagged indicated proteins. .. Cell lysates were incubated on ice for 20 min, then clarified by centrifugation at 14,000×g for 10 min. For pull-down analysis, 400 µg of protein extracts were incubated for 1 h at 4°C with 25 µl of glutathione-sepharose beads (Amersham Biosciences) to purify GST-tagged proteins.

Article Title: Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5 *
Article Snippet: Expression and Purification of GST Fusion Proteins —For producing GST fusion proteins, PCR fragments corresponding to different regions of the SCAMP2 cytoplasmic domains were inserted into a pGEX-2T bacterial expression vector (Amersham Biosciences) in-frame with the N-terminal GST tag as described previously ( ). .. GST fusion proteins were purified by incubation with reduced form glutathione-Sepharose beads (Amersham Biosciences) at 4 °C.

Article Title: RARα2 and PML-RAR similarities in the control of basal and retinoic acid induced myeloid maturation of acute myeloid leukemia cells
Article Snippet: Far-western and GST pull-down assays COS-7 cells were transfected with a pcDNA3 plasmid containing haemoagglutinin (HA)-tagged RARa 2 (pHA-RARα2 ). .. The recombinant proteins conjugated to Glutathione-sepharose beads (Amersham) were incubated with extracts of COS-7 cells transfected with pHA-RARα2 , pHA-SNAIL , pcDNA3-RARα1 , pSG5-PML-RAR or pcDNA3 containing the HA-tag (pHA ) and pcDNA3 plasmids, for 4 hours.

Article Title: Two Distinct Calmodulin Binding Sites in the Third Intracellular Loop and Carboxyl Tail of Angiotensin II (AT1A) Receptor
Article Snippet: .. Materials GST expression vector pGEX-4T-1 and Glutathione-Sepharose 4B beads were purchased from Amersham Biosciences (Piscataway, NJ). .. The E. coli BL21 gold strain was purchased from Stratagene (La Jolla, CA).

Article Title: ORF73 of Herpesvirus Saimiri, a Viral Homolog of Kaposi's Sarcoma-Associated Herpesvirus, Modulates the Two Cellular Tumor Suppressor Proteins p53 and pRb
Article Snippet: HVS ORF73 constructs were in vitro transcribed and translated with 5 μg of plasmid template and either 35 S-labeled methionine or 14 C-labeled leucine (PerkinElmer). .. For GST pulldown assays, GST, GST-p53, or GST-pRb protein was expressed in Escherichia coli DH5α cells and purified by incubation of sonicated cell lysate with glutathione-Sepharose beads (Amersham Pharmacia, Inc., Uppsala, Sweden) for 6 h at 4°C under constant rotation.

Article Title: Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap
Article Snippet: In vitro Binding Assay Plasmid pHA.DX3 DNA (0.8 μg) was used to synthesize radio-labeled DDX3 protein in vitro by utilizing a TNT T7 Coupled Reticulocyte Lysate System (Promega) in the presence of 30 μCi of [35 S]-methionine (Perkin Elmer). .. Purified GST, GST.pVIII, or GST.DDX3 fusion protein (15 μg each) was incubated individually with 20 μl of glutathione sepharose beads (GE Health Care) plus 10 μl of in vitro synthesized indicated proteins at +4°C on a nutator.

Ubiquitin Assay:

Article Title: E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue
Article Snippet: .. For the in vitro pVHL ubiquitination assay, reaction mixtures were incubated 0.5 μg of His-E1, 0.2 μg of UCP, 2 μg of pVHL and 1.25 μg Flag-ubiquitin in reaction buffer along with the ATP regeneration system at 37°C for 1 h. To precipitate the ubiquitinated pVHL, the total reaction mixtures were pulled down with Glutathione Sepharose beads or Ni-NTA agarose on a rotary shaker at 4°C for 3 h. The beads were then washed three times under reducing (1% NP40 in NET gel buffer) or non-reducing (4 M urea and 1% NP40 in NET gel buffer) conditions and resuspended in 2X SDS sample buffer under denaturing conditions. .. For the in vivo ubiquitination assay, cells were co-transfected with indicated plasmids and treated with 10 μM MG132 for 12 h before harvesting.

Recombinant:

Article Title: E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue
Article Snippet: Paragraph title: Recombinant protein extraction ... The GST-tagged proteins were then purified from the cleared lysates by affinity purification using Glutathione Sepharose beads (GE Healthcare Life Sciences).

Article Title: Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap
Article Snippet: Paragraph title: Recombinant Protein Expression and Protein Purification ... Glutathione S-transferase (GST), GST.pVIII, and GST.DDX3 fusion proteins were purified using Glutathione sepharose beads (GE Healthcare) as per the instructions of the manufacturers.

Article Title: Herpes Simplex Virus ICP27 Protein Directly Interacts with the Nuclear Pore Complex through Nup62, Inhibiting Host Nucleocytoplasmic Transport Pathways *
Article Snippet: .. GST-ICP27 , GST-Nup62, GST-RaeI, and GST alone recombinant proteins were expressed in E. coli BL21 cells and purified on glutathione-Sepharose beads (GE Healthcare) as described ( ). .. Histidine-tagged ICP27 from pET-ICP27 was expressed in BL21 DE3 and purified using Talon resin (Clontech) as described ( ).

Article Title: RARα2 and PML-RAR similarities in the control of basal and retinoic acid induced myeloid maturation of acute myeloid leukemia cells
Article Snippet: .. The recombinant proteins conjugated to Glutathione-sepharose beads (Amersham) were incubated with extracts of COS-7 cells transfected with pHA-RARα2 , pHA-SNAIL , pcDNA3-RARα1 , pSG5-PML-RAR or pcDNA3 containing the HA-tag (pHA ) and pcDNA3 plasmids, for 4 hours. .. Pulled-down proteins were subjected to WB analysis using anti-HA, anti-RARα or anti-GST antibodies.

Article Title:
Article Snippet: Paragraph title: Recombinant Protein Purification ... Cleared lysates were incubated with glutathione-Sepharose beads (Amersham Biosciences) to purify GST-tagged proteins.

In Vitro:

Article Title: Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5 *
Article Snippet: GST fusion proteins were purified by incubation with reduced form glutathione-Sepharose beads (Amersham Biosciences) at 4 °C. .. GST Pulldown —A 35 S-labeled NHE5 C-terminal domain (Gly491 -Leu896 ) was produced by in vitro transcription-translation using the T n T-coupled reticulocyte lysate system (Promega, Madison, WI) according to the manufacturer's instructions.

Article Title: ORF73 of Herpesvirus Saimiri, a Viral Homolog of Kaposi's Sarcoma-Associated Herpesvirus, Modulates the Two Cellular Tumor Suppressor Proteins p53 and pRb
Article Snippet: Paragraph title: In vitro translation and GST pulldown assays. ... For GST pulldown assays, GST, GST-p53, or GST-pRb protein was expressed in Escherichia coli DH5α cells and purified by incubation of sonicated cell lysate with glutathione-Sepharose beads (Amersham Pharmacia, Inc., Uppsala, Sweden) for 6 h at 4°C under constant rotation.

Article Title: E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue
Article Snippet: .. For the in vitro pVHL ubiquitination assay, reaction mixtures were incubated 0.5 μg of His-E1, 0.2 μg of UCP, 2 μg of pVHL and 1.25 μg Flag-ubiquitin in reaction buffer along with the ATP regeneration system at 37°C for 1 h. To precipitate the ubiquitinated pVHL, the total reaction mixtures were pulled down with Glutathione Sepharose beads or Ni-NTA agarose on a rotary shaker at 4°C for 3 h. The beads were then washed three times under reducing (1% NP40 in NET gel buffer) or non-reducing (4 M urea and 1% NP40 in NET gel buffer) conditions and resuspended in 2X SDS sample buffer under denaturing conditions. .. For the in vivo ubiquitination assay, cells were co-transfected with indicated plasmids and treated with 10 μM MG132 for 12 h before harvesting.

Article Title: Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap
Article Snippet: .. Purified GST, GST.pVIII, or GST.DDX3 fusion protein (15 μg each) was incubated individually with 20 μl of glutathione sepharose beads (GE Health Care) plus 10 μl of in vitro synthesized indicated proteins at +4°C on a nutator. .. After overnight incubation, the beads were washed three times, 10 min each with 0.1 M PBS.

Selection:

Article Title: Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5 *
Article Snippet: NHE51D4 was transfected to PC12 cells using the conventional calcium phosphate method , and cells stably expressing NHE51D4 were selected in selection media containing G418 (200 μg/ml). .. GST fusion proteins were purified by incubation with reduced form glutathione-Sepharose beads (Amersham Biosciences) at 4 °C.

Spectrophotometry:

Article Title: Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap
Article Snippet: Glutathione S-transferase (GST), GST.pVIII, and GST.DDX3 fusion proteins were purified using Glutathione sepharose beads (GE Healthcare) as per the instructions of the manufacturers. .. The concentrations of the proteins were measured by Bradford assay (Bio Rad) using Ultrospec® 3000 spectrophotometer (Pharmacia Biotech).

Produced:

Article Title: Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5 *
Article Snippet: GST fusion proteins were purified by incubation with reduced form glutathione-Sepharose beads (Amersham Biosciences) at 4 °C. .. GST Pulldown —A 35 S-labeled NHE5 C-terminal domain (Gly491 -Leu896 ) was produced by in vitro transcription-translation using the T n T-coupled reticulocyte lysate system (Promega, Madison, WI) according to the manufacturer's instructions.

Activation Assay:

Article Title: Complexes between the LKB1 tumor suppressor, STRAD?/? and MO25?/? are upstream kinases in the AMP-activated protein kinase cascade
Article Snippet: .. The E. coli lysate expressing GST-AMPKα1 was adsorbed onto glutathione-Sepharose beads (Amersham-Pharmacia) such that the final concentration of kinase after maximal activation using MgATP and AMPKK in the assay below was 1 unit in the standard kinase assay per 5 μl of beads. ..

Lysis:

Article Title: The V Protein of Tioman Virus Is Incapable of Blocking Type I Interferon Signaling in Human Cells
Article Snippet: Two days after transfection, HEK-293T cells were washed in PBS, then resuspended in lysis buffer (0.5% Nonidet P-40, 20 mM Tris–HCl at pH 8, 120 mM NaCl and 1 mM EDTA) supplemented with Complete Protease Inhibitor Cocktail (Roche). .. Cell lysates were incubated on ice for 20 min, then clarified by centrifugation at 14,000×g for 10 min. For pull-down analysis, 400 µg of protein extracts were incubated for 1 h at 4°C with 25 µl of glutathione-sepharose beads (Amersham Biosciences) to purify GST-tagged proteins.

Article Title: Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5 *
Article Snippet: Protein expression was induced by incubating transformed BL21 Escherichia coli cells with 0.2 m m isopropyl 1-thio-β- d -galactopyranoside at 37 °C for 3 h. E. coli cells were collected by centrifugation and resuspended in lysis buffer containing 1% Triton X-100 and protease inhibitor mixture (Roche Diagnostics, Laval, Canada) in PBS. .. GST fusion proteins were purified by incubation with reduced form glutathione-Sepharose beads (Amersham Biosciences) at 4 °C.

Article Title: E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue
Article Snippet: To purify GST-tagged recombinant proteins, cells were lysed in lysis buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2 HPO4 , 2 mM KH2 PO4 and 1 mM PMSF, pH 7.4) by sonication. .. The GST-tagged proteins were then purified from the cleared lysates by affinity purification using Glutathione Sepharose beads (GE Healthcare Life Sciences).

Article Title: Identification of Yin-Yang Regulators and a Phosphorylation Consensus for Male Germ Cell-Associated Kinase (MAK)-Related Kinase ▿
Article Snippet: Forty-eight hours after transfection, cells were harvested in ice-cold phosphate-buffered saline and lysed in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 2 mM EGTA, and supplemented with complete protease inhibitors [Roche], 1 mM Na3 VO4 , 1 μM microcystin LR, and 5 mM β-glycerophosphate). .. The rest of the cell lysate was incubated with glutathione-Sepharose beads (Amersham Biosciences) for 2 h at 4°C to absorb GST fusion proteins.

Chick Chorioallantoic Membrane Assay:

Article Title: Two Distinct Calmodulin Binding Sites in the Third Intracellular Loop and Carboxyl Tail of Angiotensin II (AT1A) Receptor
Article Snippet: Materials GST expression vector pGEX-4T-1 and Glutathione-Sepharose 4B beads were purchased from Amersham Biosciences (Piscataway, NJ). .. CaM and G protein Gβ antibodies were purchased from Upstate Biotechnology (Charlottesville, VA).

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    GE Healthcare glutathione sepharose beads
    Recombinant GST-Mo-MLV p12 does not associate with mitotic chromatin but is phosphorylated. (A) A representative immunoblot showing subcellular distribution of GST-p12. GST-tagged Mo-MLV p12_WT (lanes 1–3), p12_mut14 (lanes 4–6) and p12+ h CBS (lanes 7–9) were expressed in 293T cells for ~40 h. Cells were then subjected to biochemical fractionation and equivalent amounts of fractions S2-cytosolic (lanes 1, 4 and 7), S3-soluble nuclear (lanes 2, 5 and 8) and P3-chromatin pellet (lanes 3, 6 and 9) were analysed by SDS-PAGE and immunoblotting with anti-p12, anti-HSP90 (cytosolic marker) and anti-H2B (chromatin marker) antibodies. (B) Representative confocal microscopy images showing GST-p12 localisation in HeLa cells stably transduced with constructs expressing GST-tagged Mo-MLV p12_WT, p12_mut14 or p12+ h CBS. Cells were stained for p12 (anti-p12, red) and DNA (DAPI, blue). White boxes indicate mitotic cells. (C) Representative silver-stained SDS-PAGE gel (left) and immunoblot (right) of GST-p12 complexes. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT (lane 2), p12_mut14 (lane 3) or p12+ h CBS (lane 4), or GST alone (lane 1). 24 h post-transfection, cells were treated with nocodazole overnight to arrest them in mitosis and then lysed. Cell lysates were normalised on total protein concentration and GST-p12 protein complexes were precipitated with <t>glutathione-sepharose</t> beads. Bead eluates were analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-H2A, anti-H2B, anti-H3 or anti-H4 antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (D) Immunoblot showing DNA pull down assays. 293T cells were transiently-transfected with expression constructs for GST alone (top panel), GST-tagged Mo-MLV p12_WT (middle panel), or IN-HA (bottom panel) for ~40 h. DNA interacting proteins were precipitated from normalised cell lysates with cellulose beads coated with double stranded (lane 2) or single-stranded (lane 3) calf thymus DNA, and analysed by immunoblotting with anti-GST, anti-p12, or anti-IN antibodies, respectively. The arrows indicate full-length GST-p12 (~38 kDa) and IN-HA (~49 kDa) bands in the western blots. (E) GST-p12 phosphorylation. Normalised, mitotic cell lysates expressing GST-tagged Mo-MLV p12_WT (lane 3) or p12_S61A (lanes 1 and 2) were incubated with glutathione-sepharose beads. Bound proteins were separated by SDS-PAGE and the gel was sequentially stained with ProQ diamond (PQ, specifically stains phosphorylated proteins) and Sypro ruby (SR, stains all proteins) dyes. Prior to SDS-PAGE, one p12_S61A sample was treated with alkaline phosphatase (AP) for 1 h at 37°C. Band intensities were measured using a ChemiDoc imaging system and the bar chart shows PQ/SR ratios, plotted as mean ± SD of 3 technical replicates.
    Glutathione Sepharose Beads, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 1391 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Recombinant GST-Mo-MLV p12 does not associate with mitotic chromatin but is phosphorylated. (A) A representative immunoblot showing subcellular distribution of GST-p12. GST-tagged Mo-MLV p12_WT (lanes 1–3), p12_mut14 (lanes 4–6) and p12+ h CBS (lanes 7–9) were expressed in 293T cells for ~40 h. Cells were then subjected to biochemical fractionation and equivalent amounts of fractions S2-cytosolic (lanes 1, 4 and 7), S3-soluble nuclear (lanes 2, 5 and 8) and P3-chromatin pellet (lanes 3, 6 and 9) were analysed by SDS-PAGE and immunoblotting with anti-p12, anti-HSP90 (cytosolic marker) and anti-H2B (chromatin marker) antibodies. (B) Representative confocal microscopy images showing GST-p12 localisation in HeLa cells stably transduced with constructs expressing GST-tagged Mo-MLV p12_WT, p12_mut14 or p12+ h CBS. Cells were stained for p12 (anti-p12, red) and DNA (DAPI, blue). White boxes indicate mitotic cells. (C) Representative silver-stained SDS-PAGE gel (left) and immunoblot (right) of GST-p12 complexes. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT (lane 2), p12_mut14 (lane 3) or p12+ h CBS (lane 4), or GST alone (lane 1). 24 h post-transfection, cells were treated with nocodazole overnight to arrest them in mitosis and then lysed. Cell lysates were normalised on total protein concentration and GST-p12 protein complexes were precipitated with glutathione-sepharose beads. Bead eluates were analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-H2A, anti-H2B, anti-H3 or anti-H4 antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (D) Immunoblot showing DNA pull down assays. 293T cells were transiently-transfected with expression constructs for GST alone (top panel), GST-tagged Mo-MLV p12_WT (middle panel), or IN-HA (bottom panel) for ~40 h. DNA interacting proteins were precipitated from normalised cell lysates with cellulose beads coated with double stranded (lane 2) or single-stranded (lane 3) calf thymus DNA, and analysed by immunoblotting with anti-GST, anti-p12, or anti-IN antibodies, respectively. The arrows indicate full-length GST-p12 (~38 kDa) and IN-HA (~49 kDa) bands in the western blots. (E) GST-p12 phosphorylation. Normalised, mitotic cell lysates expressing GST-tagged Mo-MLV p12_WT (lane 3) or p12_S61A (lanes 1 and 2) were incubated with glutathione-sepharose beads. Bound proteins were separated by SDS-PAGE and the gel was sequentially stained with ProQ diamond (PQ, specifically stains phosphorylated proteins) and Sypro ruby (SR, stains all proteins) dyes. Prior to SDS-PAGE, one p12_S61A sample was treated with alkaline phosphatase (AP) for 1 h at 37°C. Band intensities were measured using a ChemiDoc imaging system and the bar chart shows PQ/SR ratios, plotted as mean ± SD of 3 technical replicates.

    Journal: PLoS Pathogens

    Article Title: Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis

    doi: 10.1371/journal.ppat.1007117

    Figure Lengend Snippet: Recombinant GST-Mo-MLV p12 does not associate with mitotic chromatin but is phosphorylated. (A) A representative immunoblot showing subcellular distribution of GST-p12. GST-tagged Mo-MLV p12_WT (lanes 1–3), p12_mut14 (lanes 4–6) and p12+ h CBS (lanes 7–9) were expressed in 293T cells for ~40 h. Cells were then subjected to biochemical fractionation and equivalent amounts of fractions S2-cytosolic (lanes 1, 4 and 7), S3-soluble nuclear (lanes 2, 5 and 8) and P3-chromatin pellet (lanes 3, 6 and 9) were analysed by SDS-PAGE and immunoblotting with anti-p12, anti-HSP90 (cytosolic marker) and anti-H2B (chromatin marker) antibodies. (B) Representative confocal microscopy images showing GST-p12 localisation in HeLa cells stably transduced with constructs expressing GST-tagged Mo-MLV p12_WT, p12_mut14 or p12+ h CBS. Cells were stained for p12 (anti-p12, red) and DNA (DAPI, blue). White boxes indicate mitotic cells. (C) Representative silver-stained SDS-PAGE gel (left) and immunoblot (right) of GST-p12 complexes. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT (lane 2), p12_mut14 (lane 3) or p12+ h CBS (lane 4), or GST alone (lane 1). 24 h post-transfection, cells were treated with nocodazole overnight to arrest them in mitosis and then lysed. Cell lysates were normalised on total protein concentration and GST-p12 protein complexes were precipitated with glutathione-sepharose beads. Bead eluates were analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-H2A, anti-H2B, anti-H3 or anti-H4 antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (D) Immunoblot showing DNA pull down assays. 293T cells were transiently-transfected with expression constructs for GST alone (top panel), GST-tagged Mo-MLV p12_WT (middle panel), or IN-HA (bottom panel) for ~40 h. DNA interacting proteins were precipitated from normalised cell lysates with cellulose beads coated with double stranded (lane 2) or single-stranded (lane 3) calf thymus DNA, and analysed by immunoblotting with anti-GST, anti-p12, or anti-IN antibodies, respectively. The arrows indicate full-length GST-p12 (~38 kDa) and IN-HA (~49 kDa) bands in the western blots. (E) GST-p12 phosphorylation. Normalised, mitotic cell lysates expressing GST-tagged Mo-MLV p12_WT (lane 3) or p12_S61A (lanes 1 and 2) were incubated with glutathione-sepharose beads. Bound proteins were separated by SDS-PAGE and the gel was sequentially stained with ProQ diamond (PQ, specifically stains phosphorylated proteins) and Sypro ruby (SR, stains all proteins) dyes. Prior to SDS-PAGE, one p12_S61A sample was treated with alkaline phosphatase (AP) for 1 h at 37°C. Band intensities were measured using a ChemiDoc imaging system and the bar chart shows PQ/SR ratios, plotted as mean ± SD of 3 technical replicates.

    Article Snippet: 0.5 ml aliquots of lysates at 1.5–3 mg/ml were incubated with glutathione-sepharose beads (100 μl/reaction of a 50% slurry) (GE Healthcare) for 3 h at 4°C with end-over-end rotation.

    Techniques: Recombinant, Fractionation, SDS Page, Marker, Confocal Microscopy, Stable Transfection, Transduction, Construct, Expressing, Staining, Transfection, Protein Concentration, Silver Staining, Western Blot, Incubation, Imaging

    GST-tagged Mo-MLV p12_M63I shows increased chromatin association and phosphorylation in mitosis. (A) A representative immunoblot showing subcellular distribution of GST-p12 mutants. GST-tagged GST-p12_M63I (lanes 1–3) or GST-p12+ h CBS (lanes 4–6) were expressed in 293T cells for ~40 h. Cells were then subjected to biochemical fractionation and equivalent amounts of fractions S2-cytosolic, S3-soluble nuclear and P3-chromatin pellet were analysed by SDS-PAGE and immunoblotting with anti-p12, anti-HSP90 (cytosolic marker) and anti-H2B (chromatin marker) antibodies. (B) Representative confocal microscopy images showing GST-p12 localisation in HeLa cells stably transduced with constructs expressing GST-p12_M63I and GST-p12+ h CBS. Cells were stained for p12 (anti-p12, green) and H2B (anti-H2B, red). Blue boxes indicate mitotic cells and red boxes show interphase cells. (C) Representative silver stained gel (top) and immunoblot (bottom) comparing the interaction of GST-p12_M63I and GST-p12+ h CBS with mitotic and interphase chromatin. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT, M63I or GST-p12+ h CBS for ~24 h before being treated overnight with either nocodazole (to arrest in mitosis) or aphidicolin (to block in interphase). GST-p12 protein complexes were precipitated from normalised cell lysates with glutathione-sepharose beads and analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-CLTC and anti-H2B antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (D) Quantitation of H2B pulled-down with GST-p12 from mitotic versus interphase cell lysates. Median H2B band intensities from immunoblots in (C) were measured using a Li-cor Odyssey imaging system. The increase in H2B precipitation from mitotic cell lysates relative to interphase cell lysates are plotted in the bar chart (mean ± SEM, three biological replicates). (E) GST-p12 phosphorylation in mitosis and interphase. Normalised, interphase or mitotic 293T cell lysates expressing GST-tagged Mo-MLV p12_WT, M63I or S61A were incubated with glutathione-sepharose beads. Bound proteins were separated by SDS-PAGE and the gel was sequentially stained with ProQ diamond (PQ, specifically stains phosphorylated proteins) and Sypro ruby (SR, stains all proteins) dyes. Band intensities were measured using a ChemiDoc imaging system and the bar chart shows PQ/SR ratios, plotted as mean ± SD of 3 technical replicates.

    Journal: PLoS Pathogens

    Article Title: Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis

    doi: 10.1371/journal.ppat.1007117

    Figure Lengend Snippet: GST-tagged Mo-MLV p12_M63I shows increased chromatin association and phosphorylation in mitosis. (A) A representative immunoblot showing subcellular distribution of GST-p12 mutants. GST-tagged GST-p12_M63I (lanes 1–3) or GST-p12+ h CBS (lanes 4–6) were expressed in 293T cells for ~40 h. Cells were then subjected to biochemical fractionation and equivalent amounts of fractions S2-cytosolic, S3-soluble nuclear and P3-chromatin pellet were analysed by SDS-PAGE and immunoblotting with anti-p12, anti-HSP90 (cytosolic marker) and anti-H2B (chromatin marker) antibodies. (B) Representative confocal microscopy images showing GST-p12 localisation in HeLa cells stably transduced with constructs expressing GST-p12_M63I and GST-p12+ h CBS. Cells were stained for p12 (anti-p12, green) and H2B (anti-H2B, red). Blue boxes indicate mitotic cells and red boxes show interphase cells. (C) Representative silver stained gel (top) and immunoblot (bottom) comparing the interaction of GST-p12_M63I and GST-p12+ h CBS with mitotic and interphase chromatin. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT, M63I or GST-p12+ h CBS for ~24 h before being treated overnight with either nocodazole (to arrest in mitosis) or aphidicolin (to block in interphase). GST-p12 protein complexes were precipitated from normalised cell lysates with glutathione-sepharose beads and analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-CLTC and anti-H2B antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (D) Quantitation of H2B pulled-down with GST-p12 from mitotic versus interphase cell lysates. Median H2B band intensities from immunoblots in (C) were measured using a Li-cor Odyssey imaging system. The increase in H2B precipitation from mitotic cell lysates relative to interphase cell lysates are plotted in the bar chart (mean ± SEM, three biological replicates). (E) GST-p12 phosphorylation in mitosis and interphase. Normalised, interphase or mitotic 293T cell lysates expressing GST-tagged Mo-MLV p12_WT, M63I or S61A were incubated with glutathione-sepharose beads. Bound proteins were separated by SDS-PAGE and the gel was sequentially stained with ProQ diamond (PQ, specifically stains phosphorylated proteins) and Sypro ruby (SR, stains all proteins) dyes. Band intensities were measured using a ChemiDoc imaging system and the bar chart shows PQ/SR ratios, plotted as mean ± SD of 3 technical replicates.

    Article Snippet: 0.5 ml aliquots of lysates at 1.5–3 mg/ml were incubated with glutathione-sepharose beads (100 μl/reaction of a 50% slurry) (GE Healthcare) for 3 h at 4°C with end-over-end rotation.

    Techniques: Fractionation, SDS Page, Marker, Confocal Microscopy, Stable Transfection, Transduction, Construct, Expressing, Staining, Transfection, Blocking Assay, Silver Staining, Quantitation Assay, Western Blot, Imaging, Incubation

    GST-Mo-MLV p12 recapitulates known interactions of the p12 region of Gag. Cellular proteins interacting with GST-p12 were identified using SILAC-MS. Two biological repeats (R1 and R2) were performed. (A) Schematic diagram of the SILAC-MS workflow. GST-protein complexes were isolated from normalised mitotic 293T cell lysates using glutathione-sepharose beads, pooled and subjected to LC-MS/MS analysis. (B) Identification of proteins enriched in the heavy-labelled GST-p12_WT (H) sample relative to light-labelled GST (L) sample. Log 2 (H/L) silac ratios of the set of MS hits (FDR

    Journal: PLoS Pathogens

    Article Title: Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis

    doi: 10.1371/journal.ppat.1007117

    Figure Lengend Snippet: GST-Mo-MLV p12 recapitulates known interactions of the p12 region of Gag. Cellular proteins interacting with GST-p12 were identified using SILAC-MS. Two biological repeats (R1 and R2) were performed. (A) Schematic diagram of the SILAC-MS workflow. GST-protein complexes were isolated from normalised mitotic 293T cell lysates using glutathione-sepharose beads, pooled and subjected to LC-MS/MS analysis. (B) Identification of proteins enriched in the heavy-labelled GST-p12_WT (H) sample relative to light-labelled GST (L) sample. Log 2 (H/L) silac ratios of the set of MS hits (FDR

    Article Snippet: 0.5 ml aliquots of lysates at 1.5–3 mg/ml were incubated with glutathione-sepharose beads (100 μl/reaction of a 50% slurry) (GE Healthcare) for 3 h at 4°C with end-over-end rotation.

    Techniques: Mass Spectrometry, Isolation, Liquid Chromatography with Mass Spectroscopy

    GST-p12_M63I interacts with the same chromatin-associated proteins as PFV CBS. Cellular proteins interacting with GST-p12_M63I were identified using SILAC-MS. Two biological repeats (R1 and R2) were performed. GST-p12_M63I and GST-p12_WT were transiently expressed in 293T cells cultured in light (R0/K0) or medium (R6/K4) SILAC media respectively. Cells were treated with nocodazole for mitotic enrichment and then lysed for glutathione-sepharose bead pull-down assays followed by MS. (A) Identification of proteins enriched in the light-labelled GST-p12_M63I (L) sample relative to medium-labelled GST-p12_WT (M) sample. Log 2 (L/M) silac ratios of the set of MS hits (FDR

    Journal: PLoS Pathogens

    Article Title: Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis

    doi: 10.1371/journal.ppat.1007117

    Figure Lengend Snippet: GST-p12_M63I interacts with the same chromatin-associated proteins as PFV CBS. Cellular proteins interacting with GST-p12_M63I were identified using SILAC-MS. Two biological repeats (R1 and R2) were performed. GST-p12_M63I and GST-p12_WT were transiently expressed in 293T cells cultured in light (R0/K0) or medium (R6/K4) SILAC media respectively. Cells were treated with nocodazole for mitotic enrichment and then lysed for glutathione-sepharose bead pull-down assays followed by MS. (A) Identification of proteins enriched in the light-labelled GST-p12_M63I (L) sample relative to medium-labelled GST-p12_WT (M) sample. Log 2 (L/M) silac ratios of the set of MS hits (FDR

    Article Snippet: 0.5 ml aliquots of lysates at 1.5–3 mg/ml were incubated with glutathione-sepharose beads (100 μl/reaction of a 50% slurry) (GE Healthcare) for 3 h at 4°C with end-over-end rotation.

    Techniques: Mass Spectrometry, Cell Culture

    GST-tagged Mo-MLV p12_M63I has a higher affinity for chromatin when phosphorylated. (A and B) The effect of kinase inhibitors on p12 phosphorylation (A) and chromatin association (B). 293T cells transiently-expressing GST-p12_M63I were treated overnight with nocodazole, followed by a kinase inhibitor (LiCl, roscovitine (Ros) or kenpaullone (Ken)) for 3.5 h in the presence of both nocodazole and MG132, before lysis. Normalised cell lysates were incubated with glutathione-sepharose beads, bound proteins were separated by SDS-PAGE and gels were analysed either by sequential staining with ProQ diamond (PQ) and Sypro ruby (SR) dyes (A), or by silver-staining and immunoblotting with anti-CLTC and anti-H2B antibodies. PQ/SR ratios (A) and median H2B band intensities (B) are plotted in the bar charts as mean ± SD, of three technical replicates. (C) Mitotic chromatin association of GST-p12_M63I, S61 double mutants. 293T cells transiently-expressing GST-p12_M63I +/- an S61 mutation (S61A, S61D or S61E), were treated overnight with nocodazole and analysed as in (B). (D) Infectivity of Mo-MLV VLPs carrying alterations in p12. HeLa cells were challenged with equivalent RT units of LacZ -encoding VLPs carrying Mo-MLV p12_WT or M63I, +/- S61 mutations (S61A, S61D or S61E), and infectivity was measured 72 h post-infection by detection of beta-galactosidase activity in a chemiluminescent reporter assay. The data are plotted as percentage of WT VLP infectivity (mean ± SEM of > 3 biological replicates).

    Journal: PLoS Pathogens

    Article Title: Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis

    doi: 10.1371/journal.ppat.1007117

    Figure Lengend Snippet: GST-tagged Mo-MLV p12_M63I has a higher affinity for chromatin when phosphorylated. (A and B) The effect of kinase inhibitors on p12 phosphorylation (A) and chromatin association (B). 293T cells transiently-expressing GST-p12_M63I were treated overnight with nocodazole, followed by a kinase inhibitor (LiCl, roscovitine (Ros) or kenpaullone (Ken)) for 3.5 h in the presence of both nocodazole and MG132, before lysis. Normalised cell lysates were incubated with glutathione-sepharose beads, bound proteins were separated by SDS-PAGE and gels were analysed either by sequential staining with ProQ diamond (PQ) and Sypro ruby (SR) dyes (A), or by silver-staining and immunoblotting with anti-CLTC and anti-H2B antibodies. PQ/SR ratios (A) and median H2B band intensities (B) are plotted in the bar charts as mean ± SD, of three technical replicates. (C) Mitotic chromatin association of GST-p12_M63I, S61 double mutants. 293T cells transiently-expressing GST-p12_M63I +/- an S61 mutation (S61A, S61D or S61E), were treated overnight with nocodazole and analysed as in (B). (D) Infectivity of Mo-MLV VLPs carrying alterations in p12. HeLa cells were challenged with equivalent RT units of LacZ -encoding VLPs carrying Mo-MLV p12_WT or M63I, +/- S61 mutations (S61A, S61D or S61E), and infectivity was measured 72 h post-infection by detection of beta-galactosidase activity in a chemiluminescent reporter assay. The data are plotted as percentage of WT VLP infectivity (mean ± SEM of > 3 biological replicates).

    Article Snippet: 0.5 ml aliquots of lysates at 1.5–3 mg/ml were incubated with glutathione-sepharose beads (100 μl/reaction of a 50% slurry) (GE Healthcare) for 3 h at 4°C with end-over-end rotation.

    Techniques: Expressing, Lysis, Incubation, SDS Page, Staining, Silver Staining, Mutagenesis, Infection, Activity Assay, Reporter Assay

    GST-Mo-MLV p12_M63I and other p12 orthologs associate with mitotic chromatin. (A) Representative silver stained gel (left) and immunoblot (right) showing binding of a panel of GST-p12 mutants to host proteins. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT (lane 1) and a panel of Mo-MLV p12 mutants: M63I (lane 2), G49R/E50K (lane 3), D25A/L-dom (carrying alanine substitutions of the PPPY motif as well as D25A, which disrupts clathrin binding, lane 4), p12 CTD only (lane 5) or GST-p12+ h CBS (positive control, lane 6) for ~24 h before being treated overnight with nocodazole. GST-p12 protein complexes were precipitated from normalised cell lysates with glutathione-sepharose beads and analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-CLTC, anti-WWP2, anti-H2A, anti-H2B, anti-H3 and anti-H4 antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (B) Infectivity of Mo-MLV VLPs carrying alterations in p12. HeLa cells were challenged with equivalent RT units of LacZ -encoding VLPs carrying Mo-MLV p12_WT, M63I, G49R/E50K or p12+ h CBS +/- Mut14, and infectivity was measured 72 h post-infection by detection of beta-galactosidase activity in a chemiluminescent reporter assay. The data are plotted as percentage of WT VLP infectivity (mean ± SEM of > 3 biological replicates). (C) An alignment of p12 sequences from selected gammaretroviruses. The CTD region is shaded pink. The S61 and M63 residues of Mo-MLV p12 are highlighted in red and equivalent residues at position 63 and 64 are boxed. CTD peptide sequences used in subsequent BLI assays ( Fig 9 ) are in bold. (D and E) Representative silver stained gel (top) and immunoblot (bottom) showing interaction of a panel of GST-tagged p12 orthologues (D) and GST-tagged FeLV_p12 mutants I52M and A53V (E) to chromatin associated proteins. GST-pull down assays were performed as in (A). (E) The amount of histone H2B pulled-down with GST-p12 was quantified for each sample by estimating median band intensity of immunoblots using a Li-cor Odyssey imaging system and plotted in the bar chart as mean ± SD of 3 technical replicates.

    Journal: PLoS Pathogens

    Article Title: Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis

    doi: 10.1371/journal.ppat.1007117

    Figure Lengend Snippet: GST-Mo-MLV p12_M63I and other p12 orthologs associate with mitotic chromatin. (A) Representative silver stained gel (left) and immunoblot (right) showing binding of a panel of GST-p12 mutants to host proteins. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT (lane 1) and a panel of Mo-MLV p12 mutants: M63I (lane 2), G49R/E50K (lane 3), D25A/L-dom (carrying alanine substitutions of the PPPY motif as well as D25A, which disrupts clathrin binding, lane 4), p12 CTD only (lane 5) or GST-p12+ h CBS (positive control, lane 6) for ~24 h before being treated overnight with nocodazole. GST-p12 protein complexes were precipitated from normalised cell lysates with glutathione-sepharose beads and analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-CLTC, anti-WWP2, anti-H2A, anti-H2B, anti-H3 and anti-H4 antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (B) Infectivity of Mo-MLV VLPs carrying alterations in p12. HeLa cells were challenged with equivalent RT units of LacZ -encoding VLPs carrying Mo-MLV p12_WT, M63I, G49R/E50K or p12+ h CBS +/- Mut14, and infectivity was measured 72 h post-infection by detection of beta-galactosidase activity in a chemiluminescent reporter assay. The data are plotted as percentage of WT VLP infectivity (mean ± SEM of > 3 biological replicates). (C) An alignment of p12 sequences from selected gammaretroviruses. The CTD region is shaded pink. The S61 and M63 residues of Mo-MLV p12 are highlighted in red and equivalent residues at position 63 and 64 are boxed. CTD peptide sequences used in subsequent BLI assays ( Fig 9 ) are in bold. (D and E) Representative silver stained gel (top) and immunoblot (bottom) showing interaction of a panel of GST-tagged p12 orthologues (D) and GST-tagged FeLV_p12 mutants I52M and A53V (E) to chromatin associated proteins. GST-pull down assays were performed as in (A). (E) The amount of histone H2B pulled-down with GST-p12 was quantified for each sample by estimating median band intensity of immunoblots using a Li-cor Odyssey imaging system and plotted in the bar chart as mean ± SD of 3 technical replicates.

    Article Snippet: 0.5 ml aliquots of lysates at 1.5–3 mg/ml were incubated with glutathione-sepharose beads (100 μl/reaction of a 50% slurry) (GE Healthcare) for 3 h at 4°C with end-over-end rotation.

    Techniques: Staining, Binding Assay, Transfection, Expressing, Construct, Positive Control, SDS Page, Silver Staining, Infection, Activity Assay, Reporter Assay, Western Blot, Imaging