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Effect of C-terminal truncation of LKB1 on AMPK activation in cell-free assays and ACC phosphorylation and cell cycle progress in G361 melanoma cells. A , plasmids encoding GST fusions of wild type LKB1 L and a C-terminal truncation (1–343) were co-expressed with FLAG-STRADα and myc -MO25α in HEK-293 cells and purified on <t>glutathione-Sepharose.</t> The purified products were analyzed by Western blotting using anti-GST, anti-FLAG, and anti- myc antibodies. B , a bacterially expressed GST fusion of the AMPK-α1 kinase domain was incubated with MgATP and various concentrations of GST-LKB1·FLAG-STRADα· myc -MO25α complex purified as in A , and AMPK activity was determined after 15 min. C , phosphorylation of the AMPK target, ACC, total ACC, and expression of GFP-LKB1 assessed using an anti-GFP antibody, in G361 cells co-expressing STRADα and MO25α with free GFP (control) or GFP fusions of wild type LKB1L and a C-terminally truncated mutant (1–343). D , cell cycle analysis of GFP-expressing cells treated as in Fig. 5 C , 18 h after nocodazole treatment.
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1) Product Images from "C-terminal Phosphorylation of LKB1 Is Not Required for Regulation of AMP-activated Protein Kinase, BRSK1, BRSK2, or Cell Cycle Arrest *"

Article Title: C-terminal Phosphorylation of LKB1 Is Not Required for Regulation of AMP-activated Protein Kinase, BRSK1, BRSK2, or Cell Cycle Arrest *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M806152200

Effect of C-terminal truncation of LKB1 on AMPK activation in cell-free assays and ACC phosphorylation and cell cycle progress in G361 melanoma cells. A , plasmids encoding GST fusions of wild type LKB1 L and a C-terminal truncation (1–343) were co-expressed with FLAG-STRADα and myc -MO25α in HEK-293 cells and purified on glutathione-Sepharose. The purified products were analyzed by Western blotting using anti-GST, anti-FLAG, and anti- myc antibodies. B , a bacterially expressed GST fusion of the AMPK-α1 kinase domain was incubated with MgATP and various concentrations of GST-LKB1·FLAG-STRADα· myc -MO25α complex purified as in A , and AMPK activity was determined after 15 min. C , phosphorylation of the AMPK target, ACC, total ACC, and expression of GFP-LKB1 assessed using an anti-GFP antibody, in G361 cells co-expressing STRADα and MO25α with free GFP (control) or GFP fusions of wild type LKB1L and a C-terminally truncated mutant (1–343). D , cell cycle analysis of GFP-expressing cells treated as in Fig. 5 C , 18 h after nocodazole treatment.
Figure Legend Snippet: Effect of C-terminal truncation of LKB1 on AMPK activation in cell-free assays and ACC phosphorylation and cell cycle progress in G361 melanoma cells. A , plasmids encoding GST fusions of wild type LKB1 L and a C-terminal truncation (1–343) were co-expressed with FLAG-STRADα and myc -MO25α in HEK-293 cells and purified on glutathione-Sepharose. The purified products were analyzed by Western blotting using anti-GST, anti-FLAG, and anti- myc antibodies. B , a bacterially expressed GST fusion of the AMPK-α1 kinase domain was incubated with MgATP and various concentrations of GST-LKB1·FLAG-STRADα· myc -MO25α complex purified as in A , and AMPK activity was determined after 15 min. C , phosphorylation of the AMPK target, ACC, total ACC, and expression of GFP-LKB1 assessed using an anti-GFP antibody, in G361 cells co-expressing STRADα and MO25α with free GFP (control) or GFP fusions of wild type LKB1L and a C-terminally truncated mutant (1–343). D , cell cycle analysis of GFP-expressing cells treated as in Fig. 5 C , 18 h after nocodazole treatment.

Techniques Used: Activation Assay, Purification, Western Blot, Incubation, Activity Assay, Expressing, Mutagenesis, Cell Cycle Assay

Phosphorylation and activation of AMPK, BRSK1, and BRSK2 by LKB1 variants in cell-free assays. A , purification of LKB1·STRADα·MO25α complexes from HEK-293 cells. Plasmids encoding FLAG-tagged STRADα and myc -tagged MO25α were co-expressed in HEK-293 cells with the indicated variants of GST-tagged LKB1. GST fusions were purified on glutathione-Sepharose, and the products were analyzed by Western blotting using anti-GST, anti-FLAG, or anti- myc antibodies. B –E, bacterially expressed GST fusions with the kinase domains of AMPK-α1 ( B and C ), BRSK1 ( D ), or BRSK2 ( E ) were incubated with MgATP and LKB1·STRADα·MO25α complexes (50 μg·ml –1 ) purified as in A . After 15 min the incubations were analyzed for activity of AMPK ( B ), BRSK1 ( D ), or BRSK2 ( E ) and for phosphorylation of the threonine residue equivalent to Thr-172 using anti-pT172 antibody ( C –E). WT , wild type.
Figure Legend Snippet: Phosphorylation and activation of AMPK, BRSK1, and BRSK2 by LKB1 variants in cell-free assays. A , purification of LKB1·STRADα·MO25α complexes from HEK-293 cells. Plasmids encoding FLAG-tagged STRADα and myc -tagged MO25α were co-expressed in HEK-293 cells with the indicated variants of GST-tagged LKB1. GST fusions were purified on glutathione-Sepharose, and the products were analyzed by Western blotting using anti-GST, anti-FLAG, or anti- myc antibodies. B –E, bacterially expressed GST fusions with the kinase domains of AMPK-α1 ( B and C ), BRSK1 ( D ), or BRSK2 ( E ) were incubated with MgATP and LKB1·STRADα·MO25α complexes (50 μg·ml –1 ) purified as in A . After 15 min the incubations were analyzed for activity of AMPK ( B ), BRSK1 ( D ), or BRSK2 ( E ) and for phosphorylation of the threonine residue equivalent to Thr-172 using anti-pT172 antibody ( C –E). WT , wild type.

Techniques Used: Activation Assay, Purification, Western Blot, Incubation, Activity Assay

2) Product Images from "Exportin 4 Interacts with Sox9 through the HMG Box and Inhibits the DNA Binding of Sox9"

Article Title: Exportin 4 Interacts with Sox9 through the HMG Box and Inhibits the DNA Binding of Sox9

Journal: PLoS ONE

doi: 10.1371/journal.pone.0025694

Identification of Exp4 as a major interaction partner of Sox9. (A) Silver staining of Sox9 binding proteins separated by NuPAGE. Nuclear extracts prepared from HeLa cells (HeLa NE) were incubated with (lanes 3, 4) or without FLAG-tagged Sox9 (FLAG-Sox9, lanes 1, 2). After recovery with anti-FLAG M2 antibody-conjugated agarose, the proteins were subjected to NuPAGE. The closed arrowhead indicates FLAG-Sox9, and the open arrowhead indicates the protein that was specifically recovered by FLAG-Sox9 (lane 4). (B) Nuclear extracts from U2OS cells were subjected to immunoprecipitation with anti-Sox9 antibody, and the precipitates were subjected to Western blotting analysis using anti-Exp4 antibody (right lane). Normal rabbit IgG was used as a control (middle lane). 1% of the nuclear extract was applied as a control (left lane). (C) The schematic depicts the truncated forms of Sox9 fused with GST (dark gray boxes). The numbers indicate the amino acid residues. The HMG box domain is shown as a light gray box (103–181 a.a.). (D) The upper panel shows Western blotting analysis of the protein samples co-precipitated with GST-fused truncated forms of Sox9 using an anti-Exp4 antibody. 5% of the nuclear extract was applied as a control (left lane). Numbers represent the corresponding GST-fused truncated Sox9 constructs shown in C. The lower panel shows CBB staining of NuPAGE for the GST fusion proteins used in this experiment. Numbers on the right represent the molecular weights of the marker proteins.
Figure Legend Snippet: Identification of Exp4 as a major interaction partner of Sox9. (A) Silver staining of Sox9 binding proteins separated by NuPAGE. Nuclear extracts prepared from HeLa cells (HeLa NE) were incubated with (lanes 3, 4) or without FLAG-tagged Sox9 (FLAG-Sox9, lanes 1, 2). After recovery with anti-FLAG M2 antibody-conjugated agarose, the proteins were subjected to NuPAGE. The closed arrowhead indicates FLAG-Sox9, and the open arrowhead indicates the protein that was specifically recovered by FLAG-Sox9 (lane 4). (B) Nuclear extracts from U2OS cells were subjected to immunoprecipitation with anti-Sox9 antibody, and the precipitates were subjected to Western blotting analysis using anti-Exp4 antibody (right lane). Normal rabbit IgG was used as a control (middle lane). 1% of the nuclear extract was applied as a control (left lane). (C) The schematic depicts the truncated forms of Sox9 fused with GST (dark gray boxes). The numbers indicate the amino acid residues. The HMG box domain is shown as a light gray box (103–181 a.a.). (D) The upper panel shows Western blotting analysis of the protein samples co-precipitated with GST-fused truncated forms of Sox9 using an anti-Exp4 antibody. 5% of the nuclear extract was applied as a control (left lane). Numbers represent the corresponding GST-fused truncated Sox9 constructs shown in C. The lower panel shows CBB staining of NuPAGE for the GST fusion proteins used in this experiment. Numbers on the right represent the molecular weights of the marker proteins.

Techniques Used: Silver Staining, Binding Assay, Incubation, Immunoprecipitation, Western Blot, Construct, Staining, Marker

Interaction of Exp4 with Sox family members. (A) Schematic representation of HA-tagged Sox proteins used in this study. The numbers indicate the amino acid residues. The HMG box domain is shown as a light gray box. The percentage of amino acid identity with the amino acid sequence of the HMG domain of Sox9 is given. (B) The panels show HA-affinity purification of proteins from extracts of HEK293 cells which were transiently transfected with FLAG-Exp4 and HA-Sox9, HA-Sox2, or HA-Sox11. Mock refers to the empty control plasmid. Starting materials (2% input) and bound fractions (IP, immunoprecipitation) were analyzed by NuPAGE and Western blotting. HA-tagged proteins are asterisked in the lower panels. The arrow indicates nonspecific bands. (C) The GST-fused HMG box domains of each Sox protein were separated by NuPAGE and stained with CBB (lower panel). The fusion proteins were incubated with recombinant Exp4 proteins. Proteins bound to glutathione-Sepharose were analyzed by Western blotting with anti-Exp4 antibody (upper panel). 20% input represents the control.
Figure Legend Snippet: Interaction of Exp4 with Sox family members. (A) Schematic representation of HA-tagged Sox proteins used in this study. The numbers indicate the amino acid residues. The HMG box domain is shown as a light gray box. The percentage of amino acid identity with the amino acid sequence of the HMG domain of Sox9 is given. (B) The panels show HA-affinity purification of proteins from extracts of HEK293 cells which were transiently transfected with FLAG-Exp4 and HA-Sox9, HA-Sox2, or HA-Sox11. Mock refers to the empty control plasmid. Starting materials (2% input) and bound fractions (IP, immunoprecipitation) were analyzed by NuPAGE and Western blotting. HA-tagged proteins are asterisked in the lower panels. The arrow indicates nonspecific bands. (C) The GST-fused HMG box domains of each Sox protein were separated by NuPAGE and stained with CBB (lower panel). The fusion proteins were incubated with recombinant Exp4 proteins. Proteins bound to glutathione-Sepharose were analyzed by Western blotting with anti-Exp4 antibody (upper panel). 20% input represents the control.

Techniques Used: Sequencing, Affinity Purification, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Staining, Incubation, Recombinant

3) Product Images from "Structural and Functional Consequences of Tyrosine Phosphorylation in the LRP1 Cytoplasmic Domain *"

Article Title: Structural and Functional Consequences of Tyrosine Phosphorylation in the LRP1 Cytoplasmic Domain *

Journal:

doi: 10.1074/jbc.M709514200

Phosphorylation dependence of LRP1-Shp2 interaction. A , unphosphorylated and phosphorylated GST-LRP1-CT fusion proteins immobilized on Sepharose beads were incubated with recombinant His-tagged Shp2 SH2 domains. Bound proteins were visualized by anti-His
Figure Legend Snippet: Phosphorylation dependence of LRP1-Shp2 interaction. A , unphosphorylated and phosphorylated GST-LRP1-CT fusion proteins immobilized on Sepharose beads were incubated with recombinant His-tagged Shp2 SH2 domains. Bound proteins were visualized by anti-His

Techniques Used: Incubation, Recombinant

CD Spectrum of free LRP1-CT. GST-LRP1-CT bound to glutathione-Sepharose was cleaved in solution with thrombin (1:50) in TBS, pH 7.4, 2.5 m m CaCl 2 . The free LRP1-CT was rapidly concentrated by microcolumn centrifugation before analysis. The sample
Figure Legend Snippet: CD Spectrum of free LRP1-CT. GST-LRP1-CT bound to glutathione-Sepharose was cleaved in solution with thrombin (1:50) in TBS, pH 7.4, 2.5 m m CaCl 2 . The free LRP1-CT was rapidly concentrated by microcolumn centrifugation before analysis. The sample

Techniques Used: Centrifugation

Phosphorylation at Tyr 4473 abolishes the LRP1-Snx17 interaction. A , unphosphorylated and phosphorylated GST-LRP1-CT fusion proteins immobilized on Sepharose beads were incubated with lysates of HEK293 cells expressing HA-tagged Snx17. Bound proteins
Figure Legend Snippet: Phosphorylation at Tyr 4473 abolishes the LRP1-Snx17 interaction. A , unphosphorylated and phosphorylated GST-LRP1-CT fusion proteins immobilized on Sepharose beads were incubated with lysates of HEK293 cells expressing HA-tagged Snx17. Bound proteins

Techniques Used: Incubation, Expressing

4) Product Images from "A key role for Ctf4 in coupling the MCM2-7 helicase to DNA polymerase ? within the eukaryotic replisome"

Article Title: A key role for Ctf4 in coupling the MCM2-7 helicase to DNA polymerase ? within the eukaryotic replisome

Journal:

doi: 10.1038/emboj.2009.226

The WD40 domain of Ctf4 is not essential for interaction of Ctf4 with GINS and DNA polymerase α in vivo . ( A ) Cell extracts were generated from asynchronous cultures of the indicated strains, and TAP-Sld5 isolated by immunoprecipitation with IgG-Sepharose,
Figure Legend Snippet: The WD40 domain of Ctf4 is not essential for interaction of Ctf4 with GINS and DNA polymerase α in vivo . ( A ) Cell extracts were generated from asynchronous cultures of the indicated strains, and TAP-Sld5 isolated by immunoprecipitation with IgG-Sepharose,

Techniques Used: In Vivo, Generated, Isolation, Immunoprecipitation

5) Product Images from "Glycogen Synthase Kinase-3? (GSK3?) Negatively Regulates PTTG1/Human Securin Protein Stability, and GSK3? Inactivation Correlates with Securin Accumulation in Breast Tumors *"

Article Title: Glycogen Synthase Kinase-3? (GSK3?) Negatively Regulates PTTG1/Human Securin Protein Stability, and GSK3? Inactivation Correlates with Securin Accumulation in Breast Tumors *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.232330

GSK3β interacts with securin. A , Ponceau S staining of GST fusion proteins expressed in bacteria and purified on glutathione-Sepharose columns. Mw , molecular mass markers (95, 66, 45, and 31 kDa). B , GST proteins were incubated with Nonidet P-40
Figure Legend Snippet: GSK3β interacts with securin. A , Ponceau S staining of GST fusion proteins expressed in bacteria and purified on glutathione-Sepharose columns. Mw , molecular mass markers (95, 66, 45, and 31 kDa). B , GST proteins were incubated with Nonidet P-40

Techniques Used: Staining, Purification, Incubation

6) Product Images from "Chloroplast SRP54 Was Recruited for Posttranslational Protein Transport via Complex Formation with Chloroplast SRP43 during Land Plant Evolution *"

Article Title: Chloroplast SRP54 Was Recruited for Posttranslational Protein Transport via Complex Formation with Chloroplast SRP43 during Land Plant Evolution *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.597922

Interaction analyses between various cpSRP43 and cpSRP54M constructs of Arabidopsis and Chlamydomonas . In vitro pulldown assays were performed with recombinant GST-cpSRP43 ( GST-43 ) and His-tagged cpSRP54M ( His-54M ) proteins as indicated using glutathione-Sepharose.
Figure Legend Snippet: Interaction analyses between various cpSRP43 and cpSRP54M constructs of Arabidopsis and Chlamydomonas . In vitro pulldown assays were performed with recombinant GST-cpSRP43 ( GST-43 ) and His-tagged cpSRP54M ( His-54M ) proteins as indicated using glutathione-Sepharose.

Techniques Used: Construct, In Vitro, Recombinant

7) Product Images from "Chloroplast SRP54 Was Recruited for Posttranslational Protein Transport via Complex Formation with Chloroplast SRP43 during Land Plant Evolution *"

Article Title: Chloroplast SRP54 Was Recruited for Posttranslational Protein Transport via Complex Formation with Chloroplast SRP43 during Land Plant Evolution *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.597922

Interaction analyses between various cpSRP43 and cpSRP54M constructs of Arabidopsis and Chlamydomonas . In vitro pulldown assays were performed with recombinant GST-cpSRP43 ( GST-43 ) and His-tagged cpSRP54M ( His-54M ) proteins as indicated using glutathione-Sepharose.
Figure Legend Snippet: Interaction analyses between various cpSRP43 and cpSRP54M constructs of Arabidopsis and Chlamydomonas . In vitro pulldown assays were performed with recombinant GST-cpSRP43 ( GST-43 ) and His-tagged cpSRP54M ( His-54M ) proteins as indicated using glutathione-Sepharose.

Techniques Used: Construct, In Vitro, Recombinant

8) 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

9) Product Images from "Coordinated nuclear export of 60S ribosomal subunits and NMD3 in vertebrates"

Article Title: Coordinated nuclear export of 60S ribosomal subunits and NMD3 in vertebrates

Journal: The EMBO Journal

doi: 10.1093/emboj/cdg249

Fig. 6. Export of ribosomal subunits in Xenopus oocytes requires CRM1 and Ran·GTP. ( A ) Outline of the major pathways of rRNA processing in Xenopus ), including the conversion of 12S rRNA to 6S rRNA, a precursor of 5.8S rRNA, that is matured after export to the cytoplasm (our unpublished results). (B and C) Requirement for the export receptor CRM1. ( B ) Oocytes were labeled with [ 32 P]GTP at 0 h, and treatment with LMB (400 ng/ml) was initiated at 6 h. The intracellular distributions of newly made rRNAs in control and LMB-treated oocytes were monitored at the indicated times by analysis of 0.5 oocyte equivalents of total nuclear (N) and cytoplasmic (C) RNAs in a 1.2% agarose gel. ( C ) PKI NES peptides conjugated to BSA (NES–BSA) were injected into nuclei 1 h prior to labeling with [ 32 P]GTP for 24 h, and labeled rRNAs of control and treated oocytes were analyzed as in (A). ( D ) Requirement for Ran·GTP. Oocytes were labeled with [ 32 P]GTP at 0 h, and RanT24N was injected into the cytoplasm at 24 h; rRNAs of control and treated oocytes were analyzed after 24 and 40 h of labeling, as indicated. The gel mobilities of precursor and mature rRNAs are indicated, and arrowheads show the nuclear accumulation of mature rRNAs upon inhibition of ribosomal subunit export.
Figure Legend Snippet: Fig. 6. Export of ribosomal subunits in Xenopus oocytes requires CRM1 and Ran·GTP. ( A ) Outline of the major pathways of rRNA processing in Xenopus ), including the conversion of 12S rRNA to 6S rRNA, a precursor of 5.8S rRNA, that is matured after export to the cytoplasm (our unpublished results). (B and C) Requirement for the export receptor CRM1. ( B ) Oocytes were labeled with [ 32 P]GTP at 0 h, and treatment with LMB (400 ng/ml) was initiated at 6 h. The intracellular distributions of newly made rRNAs in control and LMB-treated oocytes were monitored at the indicated times by analysis of 0.5 oocyte equivalents of total nuclear (N) and cytoplasmic (C) RNAs in a 1.2% agarose gel. ( C ) PKI NES peptides conjugated to BSA (NES–BSA) were injected into nuclei 1 h prior to labeling with [ 32 P]GTP for 24 h, and labeled rRNAs of control and treated oocytes were analyzed as in (A). ( D ) Requirement for Ran·GTP. Oocytes were labeled with [ 32 P]GTP at 0 h, and RanT24N was injected into the cytoplasm at 24 h; rRNAs of control and treated oocytes were analyzed after 24 and 40 h of labeling, as indicated. The gel mobilities of precursor and mature rRNAs are indicated, and arrowheads show the nuclear accumulation of mature rRNAs upon inhibition of ribosomal subunit export.

Techniques Used: Labeling, Agarose Gel Electrophoresis, Injection, Inhibition

10) Product Images from "Novel DNA Binding Properties of the Mcm10 Protein from Saccharomyces cerevisiae *"

Article Title: Novel DNA Binding Properties of the Mcm10 Protein from Saccharomyces cerevisiae *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.033175

Purified GST-ScMcm10 binds to duplex DNA. The GST-Mcm10 protein was purified as described under “Experimental Procedures.” A , an aliquot (200 μl) of the S-Sepharose fraction was loaded onto a linear 20 to 40% glycerol gradient
Figure Legend Snippet: Purified GST-ScMcm10 binds to duplex DNA. The GST-Mcm10 protein was purified as described under “Experimental Procedures.” A , an aliquot (200 μl) of the S-Sepharose fraction was loaded onto a linear 20 to 40% glycerol gradient

Techniques Used: Purification

11) Product Images from "Targeting Tyro3 ameliorates a model of PGRN-mutant FTLD-TDP via tau-mediated synaptic pathology"

Article Title: Targeting Tyro3 ameliorates a model of PGRN-mutant FTLD-TDP via tau-mediated synaptic pathology

Journal: Nature Communications

doi: 10.1038/s41467-018-02821-z

PGRN prevents Gas6 interaction with Tyro3. a PPI network of MARCKS retrieved from String ( http://string91.embl.de/ , http://string-db.org/ ) suggests that Tyro3 is a candidate receptor related to MARCKS. b Downstream molecules of TNFR and Tyro3 were retrieved from PPI database of the Genome Network Platform of the National Institute of Genetics ( http://genomenetwork.nig.ac.jp/index_e.html ), from which Top100 molecules with highest centrality scores (betweenness and eigenvalue) were listed. 10 molecules are shared by Top100 lists of TNFR and Tyro3, in which Shc and Grb2 are identified as critical signal mediators. c Interaction of Gas6 with the plasma membrane in primary cortical neurons expressing Tyro3-TurboGFP, and inhibition of this interaction by PGRN. d SPR analysis revealed Gas6 binding to Tyro3 and the inhibition of this interaction by PGRN (left panel). BDNF did not inhibit binding of Gas6 to Tyro3 (right panel). e , f , g Affinities of Gas6, PGRN, and BDNF to Tyro3 were evaluated by SPR. Gas6, but not PGRN or BDNF, bound Tyro3 with high affinity. h PGRN bound Gas6 at high affinity. i BDNF did not bind to Gas6. j Pre-incubation of Gas6 with PGRN decreased the affinity of Gas6 for Tyro3. k Pull-down assay revealed direct interaction between PGRN and Gas6. His-tagged proteins were pulled down by Ni-NTA-agarose, and GST-tagged proteins were pulled down by glutathione sepharose
Figure Legend Snippet: PGRN prevents Gas6 interaction with Tyro3. a PPI network of MARCKS retrieved from String ( http://string91.embl.de/ , http://string-db.org/ ) suggests that Tyro3 is a candidate receptor related to MARCKS. b Downstream molecules of TNFR and Tyro3 were retrieved from PPI database of the Genome Network Platform of the National Institute of Genetics ( http://genomenetwork.nig.ac.jp/index_e.html ), from which Top100 molecules with highest centrality scores (betweenness and eigenvalue) were listed. 10 molecules are shared by Top100 lists of TNFR and Tyro3, in which Shc and Grb2 are identified as critical signal mediators. c Interaction of Gas6 with the plasma membrane in primary cortical neurons expressing Tyro3-TurboGFP, and inhibition of this interaction by PGRN. d SPR analysis revealed Gas6 binding to Tyro3 and the inhibition of this interaction by PGRN (left panel). BDNF did not inhibit binding of Gas6 to Tyro3 (right panel). e , f , g Affinities of Gas6, PGRN, and BDNF to Tyro3 were evaluated by SPR. Gas6, but not PGRN or BDNF, bound Tyro3 with high affinity. h PGRN bound Gas6 at high affinity. i BDNF did not bind to Gas6. j Pre-incubation of Gas6 with PGRN decreased the affinity of Gas6 for Tyro3. k Pull-down assay revealed direct interaction between PGRN and Gas6. His-tagged proteins were pulled down by Ni-NTA-agarose, and GST-tagged proteins were pulled down by glutathione sepharose

Techniques Used: Expressing, Inhibition, SPR Assay, Binding Assay, Incubation, Pull Down Assay

12) Product Images from "Targeting of Protein Phosphatases PP2A and PP2B to the C-terminus of the L-type Calcium Channel Cav1.2"

Article Title: Targeting of Protein Phosphatases PP2A and PP2B to the C-terminus of the L-type Calcium Channel Cav1.2

Journal: Biochemistry

doi: 10.1021/bi101018c

PP2A/C and PP2B do not compete for CT-8 binding. Glutathione Sepharose was sequentially incubated with GST-CT-8, then 0, 1.1, or 5.5 μg PP2A, and finally 0.2 μg PP2B. Representative immunoblots (left panels) illustrate the relative amount
Figure Legend Snippet: PP2A/C and PP2B do not compete for CT-8 binding. Glutathione Sepharose was sequentially incubated with GST-CT-8, then 0, 1.1, or 5.5 μg PP2A, and finally 0.2 μg PP2B. Representative immunoblots (left panels) illustrate the relative amount

Techniques Used: Binding Assay, Incubation, Western Blot

13) Product Images from "Scar/WAVE-1, a Wiskott-Aldrich syndrome protein, assembles an actin-associated multi-kinase scaffold"

Article Title: Scar/WAVE-1, a Wiskott-Aldrich syndrome protein, assembles an actin-associated multi-kinase scaffold

Journal: The EMBO Journal

doi: 10.1093/emboj/19.17.4589

Fig. 4. Mapping the Abl-binding domain. ( A ) A schematic representation of recombinant His-tagged WAVE-1 fusion fragments used in mapping studies is presented. Fragments that interact with the GST–SH3 domain from Abl (filled boxes) and fragments negative for binding (open boxes) are indicated. The first and last residues in each fragment of WAVE-1 are numbered. ( B ) Recombinant fragments of WAVE-1 were incubated with GST–Abl SH3 fusion protein that was immobilized to glutathione–Sepharose for 1 h at 4°C. Bound proteins were detected by immunoblotting using a monoclonal antibody to the histidine tag present on the WAVE fragments. ( C ) A control immunoblot indicating the expression levels of each His-tagged WAVE-1 fragment loaded onto the GST–Abl SH3 domain column. WAVE-1 fragments were detected by immunoblotting using a monoclonal antibody to the histidine tag. Molecular weight standards are indicated. Representative examples of three individual experiments are presented.
Figure Legend Snippet: Fig. 4. Mapping the Abl-binding domain. ( A ) A schematic representation of recombinant His-tagged WAVE-1 fusion fragments used in mapping studies is presented. Fragments that interact with the GST–SH3 domain from Abl (filled boxes) and fragments negative for binding (open boxes) are indicated. The first and last residues in each fragment of WAVE-1 are numbered. ( B ) Recombinant fragments of WAVE-1 were incubated with GST–Abl SH3 fusion protein that was immobilized to glutathione–Sepharose for 1 h at 4°C. Bound proteins were detected by immunoblotting using a monoclonal antibody to the histidine tag present on the WAVE fragments. ( C ) A control immunoblot indicating the expression levels of each His-tagged WAVE-1 fragment loaded onto the GST–Abl SH3 domain column. WAVE-1 fragments were detected by immunoblotting using a monoclonal antibody to the histidine tag. Molecular weight standards are indicated. Representative examples of three individual experiments are presented.

Techniques Used: Binding Assay, Recombinant, Incubation, Expressing, Molecular Weight

14) Product Images from "Ku-0063794 is a specific inhibitor of the mammalian target of rapamycin (mTOR)"

Article Title: Ku-0063794 is a specific inhibitor of the mammalian target of rapamycin (mTOR)

Journal: Biochemical Journal

doi: 10.1042/BJ20090489

Ku-0063794 suppresses hydrophobic motif phosphorylation and activation of SGK1 but not RSK ( A ) HEK-293 cells were transfected with a DNA construct encoding GST–SGK1 (full-length enzyme). Cells were cultured in the presence of 10% foetal bovine serum in order to maintain PI3K pathway activity. At 36 h post-transfection, cells were treated for 30 min in the absence or presence of the indicated concentrations of Ku-0063794 or PI-103. Cells were lysed, SGK1 was affinity-purified on glutathione–Sepharose and catalytic activity was assessed employing the Crosstide substrate. Each bar represents the mean specific activity±S.E.M. from three different samples, with each sample assayed in duplicate. Affinity purified SGK1 was also subjected to immunoblotting with an anti-GST antibody (SGK1-Total) as well as an anti- Ser 422 phosphospecific antibody (S422-P). Cell lysates were also analysed by immunoblotting with the indicated non-SGK antibodies. ( B and C ) HeLa cells or the indicated wild-type (wt) or knockout (ko) MEFs were cultured in the presence of 10% serum, then treated for 30 min in the absence or presence of the indicated concentrations of inhibitors. Cells were lysed and extracts were analysed by immunoblotting with the indicated antibodies. Immunoblots are representative of three different experiments. ( D ) HEK-293 cells were deprived of serum for 16 h, treated for 30 min in the absence or presence of 1 μM Ku-0063794 or 0.2 μM PD 0325901 then stimulated with 400 ng/ml of PMA for 15 min. RSK was immunoprecipitated with an antibody recognizing all isoforms and catalytic activity assessed employing the Crosstide substrate. Each bar represents the mean specific activity±S.E.M. from two different samples, with each sample assayed in duplicate. Cell lysates were also analysed by immunoblotting with the indicated antibodies. P, phosphorylated.
Figure Legend Snippet: Ku-0063794 suppresses hydrophobic motif phosphorylation and activation of SGK1 but not RSK ( A ) HEK-293 cells were transfected with a DNA construct encoding GST–SGK1 (full-length enzyme). Cells were cultured in the presence of 10% foetal bovine serum in order to maintain PI3K pathway activity. At 36 h post-transfection, cells were treated for 30 min in the absence or presence of the indicated concentrations of Ku-0063794 or PI-103. Cells were lysed, SGK1 was affinity-purified on glutathione–Sepharose and catalytic activity was assessed employing the Crosstide substrate. Each bar represents the mean specific activity±S.E.M. from three different samples, with each sample assayed in duplicate. Affinity purified SGK1 was also subjected to immunoblotting with an anti-GST antibody (SGK1-Total) as well as an anti- Ser 422 phosphospecific antibody (S422-P). Cell lysates were also analysed by immunoblotting with the indicated non-SGK antibodies. ( B and C ) HeLa cells or the indicated wild-type (wt) or knockout (ko) MEFs were cultured in the presence of 10% serum, then treated for 30 min in the absence or presence of the indicated concentrations of inhibitors. Cells were lysed and extracts were analysed by immunoblotting with the indicated antibodies. Immunoblots are representative of three different experiments. ( D ) HEK-293 cells were deprived of serum for 16 h, treated for 30 min in the absence or presence of 1 μM Ku-0063794 or 0.2 μM PD 0325901 then stimulated with 400 ng/ml of PMA for 15 min. RSK was immunoprecipitated with an antibody recognizing all isoforms and catalytic activity assessed employing the Crosstide substrate. Each bar represents the mean specific activity±S.E.M. from two different samples, with each sample assayed in duplicate. Cell lysates were also analysed by immunoblotting with the indicated antibodies. P, phosphorylated.

Techniques Used: Activation Assay, Transfection, Construct, Cell Culture, Activity Assay, Affinity Purification, Knock-Out, Western Blot, Immunoprecipitation

15) Product Images from "Emp47p and Its Close Homolog Emp46p Have a Tyrosine-containing Endoplasmic Reticulum Exit Signal and Function in Glycoprotein Secretion in Saccharomyces cerevisiae"

Article Title: Emp47p and Its Close Homolog Emp46p Have a Tyrosine-containing Endoplasmic Reticulum Exit Signal and Function in Glycoprotein Secretion in Saccharomyces cerevisiae

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E02-01-0027

Secretion of glycoproteins by emp46/47 deletion strains. Cells were labeled with [ 35 S]methionine for 10 min and were chased for 30 min. Media were collected and incubated with ConA-Sepharose to isolate extracellular glycoproteins. Proteins were separated by SDS-PAGE, and labeled proteins were detected by autoradiography. ● in A and ○ in B mark glycoproteins secreted inefficiently by mutant cells. Arrowheads designate glycoproteins secreted underglycosylated by the emp47 Δ cells. (B) Low molecular weight region with increased samples.
Figure Legend Snippet: Secretion of glycoproteins by emp46/47 deletion strains. Cells were labeled with [ 35 S]methionine for 10 min and were chased for 30 min. Media were collected and incubated with ConA-Sepharose to isolate extracellular glycoproteins. Proteins were separated by SDS-PAGE, and labeled proteins were detected by autoradiography. ● in A and ○ in B mark glycoproteins secreted inefficiently by mutant cells. Arrowheads designate glycoproteins secreted underglycosylated by the emp47 Δ cells. (B) Low molecular weight region with increased samples.

Techniques Used: Labeling, Incubation, SDS Page, Autoradiography, Mutagenesis, Molecular Weight

Binding of COPI (Ret1p and Sec21p) and COPII (Sec23p) to the C-terminal tail of Emp46p. GST fusion proteins comprising the C-terminal tail of Emp47p or Emp46p in either wild-type form (WT) or with a mutation as indicated were immobilized on glutathione-Sepharose beads and incubated with whole-cell lysate for 2 h at 4°C. The beads were washed and bound proteins were eluted from the beads and analyzed by SDS-PAGE and imm unoblotting with anticoatomer, anti-Sec21p, or anti-Sec23p.
Figure Legend Snippet: Binding of COPI (Ret1p and Sec21p) and COPII (Sec23p) to the C-terminal tail of Emp46p. GST fusion proteins comprising the C-terminal tail of Emp47p or Emp46p in either wild-type form (WT) or with a mutation as indicated were immobilized on glutathione-Sepharose beads and incubated with whole-cell lysate for 2 h at 4°C. The beads were washed and bound proteins were eluted from the beads and analyzed by SDS-PAGE and imm unoblotting with anticoatomer, anti-Sec21p, or anti-Sec23p.

Techniques Used: Binding Assay, Mutagenesis, Incubation, SDS Page

16) Product Images from "Epstein-Barr virus glycoprotein gM can interact with the cellular protein p32 and knockdown of p32 impairs virus"

Article Title: Epstein-Barr virus glycoprotein gM can interact with the cellular protein p32 and knockdown of p32 impairs virus

Journal: Virology

doi: 10.1016/j.virol.2015.12.019

Proteins pulled down by GST or GST-gM. SDS-PAGE and autoradiography of extracts of induced (I) or uninduced (U) Akata cells labeled with [ 3 H]-leucine and pulled down with GST or GST-gM bound to glutathione-Sepharose.
Figure Legend Snippet: Proteins pulled down by GST or GST-gM. SDS-PAGE and autoradiography of extracts of induced (I) or uninduced (U) Akata cells labeled with [ 3 H]-leucine and pulled down with GST or GST-gM bound to glutathione-Sepharose.

Techniques Used: SDS Page, Autoradiography, Labeling

17) Product Images from "Discovery of the Elusive Leptin in Birds: Identification of Several 'Missing Links' in the Evolution of Leptin and Its Receptor"

Article Title: Discovery of the Elusive Leptin in Birds: Identification of Several 'Missing Links' in the Evolution of Leptin and Its Receptor

Journal: PLoS ONE

doi: 10.1371/journal.pone.0092751

Purification of Peregrine falcon lepin (pfleptin). A) Coomassie stain following 15% Tris-Tricine SDS PAGE. Control lysate and lysate of the pJ414 pfleptin expressing cells were previously passed over Ni-Sepharose and dialyzed into 125 mM NaCl. 15 μL of each sample were loaded onto the gel. The pfleptin protein with tag is 17 kDa. B) Western blot of A following transfer to membrane and probed with an anti-His primary antibody. C) Glutathione pull down of the pGEX4T-chLepR(227–628) which can be seen at 71 kDa. Beads were then incubated with either the control lysate or the pfleptin lysate. This resulted in concentrating the pfleptin protein at 17 kDa. D) Luciferase assays showing activation of the STAT pathway of chicken leptin receptor transfected CHO cells treated with concentrated peregrine falcon leptin (n = 2). Error bars represent the standard error of the mean and * represents a p-value ≤ 0.005 between samples. No significance was seen between the two concentrations of leptin treatment suggesting maximum saturation in the assay was reached.
Figure Legend Snippet: Purification of Peregrine falcon lepin (pfleptin). A) Coomassie stain following 15% Tris-Tricine SDS PAGE. Control lysate and lysate of the pJ414 pfleptin expressing cells were previously passed over Ni-Sepharose and dialyzed into 125 mM NaCl. 15 μL of each sample were loaded onto the gel. The pfleptin protein with tag is 17 kDa. B) Western blot of A following transfer to membrane and probed with an anti-His primary antibody. C) Glutathione pull down of the pGEX4T-chLepR(227–628) which can be seen at 71 kDa. Beads were then incubated with either the control lysate or the pfleptin lysate. This resulted in concentrating the pfleptin protein at 17 kDa. D) Luciferase assays showing activation of the STAT pathway of chicken leptin receptor transfected CHO cells treated with concentrated peregrine falcon leptin (n = 2). Error bars represent the standard error of the mean and * represents a p-value ≤ 0.005 between samples. No significance was seen between the two concentrations of leptin treatment suggesting maximum saturation in the assay was reached.

Techniques Used: Purification, Staining, SDS Page, Expressing, Western Blot, Incubation, Luciferase, Activation Assay, Transfection

18) Product Images from "Interaction between the cellular E3 ubiquitin ligase SIAH-1 and the viral immediate-early protein ICP0 enables efficient replication of Herpes Simplex Virus type 2 in vivo"

Article Title: Interaction between the cellular E3 ubiquitin ligase SIAH-1 and the viral immediate-early protein ICP0 enables efficient replication of Herpes Simplex Virus type 2 in vivo

Journal: PLoS ONE

doi: 10.1371/journal.pone.0201880

ICP0 interacts with SIAH-1 via two minimal interaction motifs. ( A ) Schematic representation of HSV-2 ICP0 indicating the position of the RING domain (yellow), the USP7 interaction domain (blue), the nuclear localization signal (brown) and the two SIAH interaction motifs VxP1 and VxP2 (red). The primary amino acid sequence surrounding the predicted SIAH binding motifs is depicted together with the consensus motif and the position of the inactivating NxN mutation. ( B ) HEK293T cells were transfected with plasmids encoding GFP-tagged ICP0 and its mutants and the cell lysates were incubated with GST or GST-SIAH-1-loaded glutathione sepharose beads. The upper SDS-PAGE gel shows a Coomassie staining of the respective input control (lysate) and the eluates from the GST or GST-SIAH-1 beads. Below, a contrast enhanced section of the gel with putative ICP0 bands indicated by asterisks. ICP0 was detected by Western blotting using an antibody against the C-terminal GFP tag. Size markers in kDa. ( C ) HEK293T cells were transfected with plasmids encoding GFP-tagged ICP0 and its mutants and HA-tagged SIAH-1. The ICP0-GFP proteins were immunoprecipitated from the lysates, ICP0 and SIAH-1 were detected by SDS-PAGE and Western blotting using antibodies against the GFP and HA-tags. ( D ) HEK293T cells were transfected with plasmids encoding the inactive mutant HA-SIAH-1 C44S and GFP-ICP0 ΔRING or GFP-ICP0 ΔRING/NxN1/2 as indicated. Immunoprecipitation from the cell lysates was performed using control mouse IgG or anti-SIAH-1. ICP0 and SIAH-1 were detected by SDS-PAGE and Western blotting using antibodies against SIAH-1 or the GFP-tag. The lower panel shows the analysis of the RIPA buffer-insoluble pellet after cell lysis.
Figure Legend Snippet: ICP0 interacts with SIAH-1 via two minimal interaction motifs. ( A ) Schematic representation of HSV-2 ICP0 indicating the position of the RING domain (yellow), the USP7 interaction domain (blue), the nuclear localization signal (brown) and the two SIAH interaction motifs VxP1 and VxP2 (red). The primary amino acid sequence surrounding the predicted SIAH binding motifs is depicted together with the consensus motif and the position of the inactivating NxN mutation. ( B ) HEK293T cells were transfected with plasmids encoding GFP-tagged ICP0 and its mutants and the cell lysates were incubated with GST or GST-SIAH-1-loaded glutathione sepharose beads. The upper SDS-PAGE gel shows a Coomassie staining of the respective input control (lysate) and the eluates from the GST or GST-SIAH-1 beads. Below, a contrast enhanced section of the gel with putative ICP0 bands indicated by asterisks. ICP0 was detected by Western blotting using an antibody against the C-terminal GFP tag. Size markers in kDa. ( C ) HEK293T cells were transfected with plasmids encoding GFP-tagged ICP0 and its mutants and HA-tagged SIAH-1. The ICP0-GFP proteins were immunoprecipitated from the lysates, ICP0 and SIAH-1 were detected by SDS-PAGE and Western blotting using antibodies against the GFP and HA-tags. ( D ) HEK293T cells were transfected with plasmids encoding the inactive mutant HA-SIAH-1 C44S and GFP-ICP0 ΔRING or GFP-ICP0 ΔRING/NxN1/2 as indicated. Immunoprecipitation from the cell lysates was performed using control mouse IgG or anti-SIAH-1. ICP0 and SIAH-1 were detected by SDS-PAGE and Western blotting using antibodies against SIAH-1 or the GFP-tag. The lower panel shows the analysis of the RIPA buffer-insoluble pellet after cell lysis.

Techniques Used: Sequencing, Binding Assay, Mutagenesis, Transfection, Incubation, SDS Page, Staining, Western Blot, Immunoprecipitation, Lysis

Virally expressed ICP0 NxN1/2 does not bind to SIAH-1. ( A ) U2OS cells were infected for 48 h with the indicated mutants and HSV-2 strain MS (MS wt) at an MOI 0.01 pfu/cell. Cell lysates were incubated with GST-SIAH-1-loaded glutathione sepharose beads. Eluates were analyzed by SDS-PAGE and Western blotting using antibodies directed against HSV-2 ICP0 and GFP. ( B ) Input controls (10%) of the GST-pulldown were analyzed as before using ICP0-specific antibody.
Figure Legend Snippet: Virally expressed ICP0 NxN1/2 does not bind to SIAH-1. ( A ) U2OS cells were infected for 48 h with the indicated mutants and HSV-2 strain MS (MS wt) at an MOI 0.01 pfu/cell. Cell lysates were incubated with GST-SIAH-1-loaded glutathione sepharose beads. Eluates were analyzed by SDS-PAGE and Western blotting using antibodies directed against HSV-2 ICP0 and GFP. ( B ) Input controls (10%) of the GST-pulldown were analyzed as before using ICP0-specific antibody.

Techniques Used: Infection, Mass Spectrometry, Incubation, SDS Page, Western Blot

19) Product Images from "TANGO1 recruits Sec16 to coordinately organize ER exit sites for efficient secretion"

Article Title: TANGO1 recruits Sec16 to coordinately organize ER exit sites for efficient secretion

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201703084

TANGO1 interacts with Sec16. (A) 293T cells were transfected with TANGO1S-HA or TANGO1L-HA with FLAG-Sec16 constructs as indicated. Cell lysates were immunoprecipitated with anti-FLAG antibody and eluted with a FLAG peptide. Eluates were then subjected to SDS-PAGE followed by Western blotting with anti-FLAG and anti-HA antibodies. (B) Schematic representation of human Sec16A domain organization. CCD, central conserved domain; CTCD, C-terminal conserved domain. (C) 293T cells were transfected with FLAG-Sec16 (1–550 aa), FLAG-Sec16 (551–1,100 aa), FLAG-Sec16 (1,101–1,600 aa), FLAG-Sec16 (1,601–1,890 aa), or FLAG-Sec16 (1,891–2,332 aa) with TANGO1S-HA constructs as indicated. Cell lysates were immunoprecipitated with anti-FLAG antibody and eluted with a FLAG peptide. Eluates were then subjected to SDS-PAGE followed by Western blotting with anti-FLAG and anti-HA antibodies. (D) Recombinant GST, GST-tagged TANGO1-coil1 (1,211–1,440 aa), GST-tagged TANGO1-coil2 (1,441–1,650 aa), or GST-tagged TANGO1-PRD (1,651–1,907 aa) were immobilized to glutathione Sepharose resin and incubated with FLAG-Sec16 (1,101–1,600 aa). Resins were washed and eluted with glutathione. Eluted proteins were subjected to SDS-PAGE followed by Western blotting with anti-FLAG and anti-GST antibodies. (E and F) GST, GST-tagged TANGO1-PRD (1,651–1,907 aa), GST-tagged TANGO1-PRDΔ45 (1,651–1,862 aa), GST-tagged TANGO1-PRDΔ60 (1,651–1,847 aa), or GST-tagged TANGO1-PRDΔ120 (1,651–1,787 aa) were immobilized to glutathione Sepharose resin and incubated with FLAG-Sec16 (1,101–1,600 aa; E) or FLAG-Sec23A (F). Resins were washed and eluted with glutathione. Eluted proteins were subject to SDS-PAGE followed by Western blotting with anti-FLAG and anti-GST antibodies.
Figure Legend Snippet: TANGO1 interacts with Sec16. (A) 293T cells were transfected with TANGO1S-HA or TANGO1L-HA with FLAG-Sec16 constructs as indicated. Cell lysates were immunoprecipitated with anti-FLAG antibody and eluted with a FLAG peptide. Eluates were then subjected to SDS-PAGE followed by Western blotting with anti-FLAG and anti-HA antibodies. (B) Schematic representation of human Sec16A domain organization. CCD, central conserved domain; CTCD, C-terminal conserved domain. (C) 293T cells were transfected with FLAG-Sec16 (1–550 aa), FLAG-Sec16 (551–1,100 aa), FLAG-Sec16 (1,101–1,600 aa), FLAG-Sec16 (1,601–1,890 aa), or FLAG-Sec16 (1,891–2,332 aa) with TANGO1S-HA constructs as indicated. Cell lysates were immunoprecipitated with anti-FLAG antibody and eluted with a FLAG peptide. Eluates were then subjected to SDS-PAGE followed by Western blotting with anti-FLAG and anti-HA antibodies. (D) Recombinant GST, GST-tagged TANGO1-coil1 (1,211–1,440 aa), GST-tagged TANGO1-coil2 (1,441–1,650 aa), or GST-tagged TANGO1-PRD (1,651–1,907 aa) were immobilized to glutathione Sepharose resin and incubated with FLAG-Sec16 (1,101–1,600 aa). Resins were washed and eluted with glutathione. Eluted proteins were subjected to SDS-PAGE followed by Western blotting with anti-FLAG and anti-GST antibodies. (E and F) GST, GST-tagged TANGO1-PRD (1,651–1,907 aa), GST-tagged TANGO1-PRDΔ45 (1,651–1,862 aa), GST-tagged TANGO1-PRDΔ60 (1,651–1,847 aa), or GST-tagged TANGO1-PRDΔ120 (1,651–1,787 aa) were immobilized to glutathione Sepharose resin and incubated with FLAG-Sec16 (1,101–1,600 aa; E) or FLAG-Sec23A (F). Resins were washed and eluted with glutathione. Eluted proteins were subject to SDS-PAGE followed by Western blotting with anti-FLAG and anti-GST antibodies.

Techniques Used: Transfection, Construct, Immunoprecipitation, SDS Page, Western Blot, Recombinant, Incubation

20) Product Images from "The VirD2 pilot protein of Agrobacterium-transferred DNA interacts with the TATA box-binding protein and a nuclear protein kinase in plants"

Article Title: The VirD2 pilot protein of Agrobacterium-transferred DNA interacts with the TATA box-binding protein and a nuclear protein kinase in plants

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

doi: 10.1073/pnas.1733208100

Molecular interactions among VirD2, TBP, and RNA polymerase II CTD. ( A ) SDS/PAGE analysis of in vitro protein interactions shows that MBP-CTD binds TBP, but not VirD2, whereas VirD2 recruits TBP. ( B )( Upper ) His 6 -TBP forms a salt-resistant complex with GST-VirD2 but does not bind specifically to control glutathione-Sepharose and GST-loaded beads. ( Lower ) The N-terminal VirD2 domain is necessary for TBP binding. GST-VirD2ΔNT, carrying an N-terminal VirD2 deletion of 266 aa, and the control GST protein show only trace levels of TBP binding. GST-VirD2 and GST-VirD2ΔCT, carrying a C-terminal VirD2 deletion, show strong interaction with TBP. ( C ) Transient expression of HA-VirD2 by Agrobacterium transformation in Arabidopsis cells. Multiple forms of HA-VirD2 protein are observed by immunoblotting with anti-HA and anti-VirD2 IgGs, as well as by anti-HA immunoprecipitation (αHA-IP), followed by Western blotting with anti-VirD2 IgG. ( D ) ( Left ) Detection of TBP in wild-type cells by Western blotting with anti-TBP IgG. ( Right ) Detection of TPB after immunoprecipitation of protein extracts from wild-type cells (wt) and HA-VirD2-expressing Agrobacterium -transformed Arabidopsis cells with anti-TBP Ab.
Figure Legend Snippet: Molecular interactions among VirD2, TBP, and RNA polymerase II CTD. ( A ) SDS/PAGE analysis of in vitro protein interactions shows that MBP-CTD binds TBP, but not VirD2, whereas VirD2 recruits TBP. ( B )( Upper ) His 6 -TBP forms a salt-resistant complex with GST-VirD2 but does not bind specifically to control glutathione-Sepharose and GST-loaded beads. ( Lower ) The N-terminal VirD2 domain is necessary for TBP binding. GST-VirD2ΔNT, carrying an N-terminal VirD2 deletion of 266 aa, and the control GST protein show only trace levels of TBP binding. GST-VirD2 and GST-VirD2ΔCT, carrying a C-terminal VirD2 deletion, show strong interaction with TBP. ( C ) Transient expression of HA-VirD2 by Agrobacterium transformation in Arabidopsis cells. Multiple forms of HA-VirD2 protein are observed by immunoblotting with anti-HA and anti-VirD2 IgGs, as well as by anti-HA immunoprecipitation (αHA-IP), followed by Western blotting with anti-VirD2 IgG. ( D ) ( Left ) Detection of TBP in wild-type cells by Western blotting with anti-TBP IgG. ( Right ) Detection of TPB after immunoprecipitation of protein extracts from wild-type cells (wt) and HA-VirD2-expressing Agrobacterium -transformed Arabidopsis cells with anti-TBP Ab.

Techniques Used: SDS Page, In Vitro, Binding Assay, Expressing, Transformation Assay, Immunoprecipitation, Western Blot

21) Product Images from "CHD8 Is an ATP-Dependent Chromatin Remodeling Factor That Regulates β-Catenin Target Genes "

Article Title: CHD8 Is an ATP-Dependent Chromatin Remodeling Factor That Regulates β-Catenin Target Genes

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00322-08

CHD8 is a component of a large complex in HeLa nuclear extracts. (A) HeLa nuclear extract was fractionated by phosphocellulose (P11) chromatography utilizing stepwise elution with the indicated KCl concentrations (0.1 M, 0.3 M, 0.5 M, 1.0 M). Western blot analysis was performed using affinity-purified anti-CHD8 antibodies. The 0.5 M P11 fraction was further fractionated by DEAE-Sephacel chromatography and eluted stepwise with 0.35 M KCl. Samples were further resolved by chromatography on a Superose 6 HR 10/30 column. Western blotting was performed using affinity-purified anti-CHD8 antibodies. Arrows (bottom) indicate the elution position of thyroglobulin (670 kDa) and the void volume of the column (∼2 MDa). (B) Immunoprecipitation of CHD8. HeLa nuclear extract was incubated with anti-CHD8 antibodies (α-CHD8) or normal rabbit IgG (α-Control) cross-linked to protein A agarose. After extensive washes to remove nonspecific interacting proteins, immunoprecipitates were subjected to SDS-PAGE, and proteins were visualized by Colloidal Blue staining. (C) 293 cells were transfected with Flag-WDR5 or the control vector. Immunoprecipitations (IP) were performed with anti-Flag M2 antibodies. After washing, purified samples were subjected to SDS-PAGE followed by Western blot analysis (WB) using the antibodies indicated. (D) Recombinant CHD8 was incubated with recombinant GST or GST-WDR5 as indicated at the top. After washing, glutathione-agarose-purified samples were subjected to SDS-PAGE. The top of the gel was subjected to Western blot analysis using anti-CHD8 antibodies. The bottom of the gel was analyzed by Coomassie staining.
Figure Legend Snippet: CHD8 is a component of a large complex in HeLa nuclear extracts. (A) HeLa nuclear extract was fractionated by phosphocellulose (P11) chromatography utilizing stepwise elution with the indicated KCl concentrations (0.1 M, 0.3 M, 0.5 M, 1.0 M). Western blot analysis was performed using affinity-purified anti-CHD8 antibodies. The 0.5 M P11 fraction was further fractionated by DEAE-Sephacel chromatography and eluted stepwise with 0.35 M KCl. Samples were further resolved by chromatography on a Superose 6 HR 10/30 column. Western blotting was performed using affinity-purified anti-CHD8 antibodies. Arrows (bottom) indicate the elution position of thyroglobulin (670 kDa) and the void volume of the column (∼2 MDa). (B) Immunoprecipitation of CHD8. HeLa nuclear extract was incubated with anti-CHD8 antibodies (α-CHD8) or normal rabbit IgG (α-Control) cross-linked to protein A agarose. After extensive washes to remove nonspecific interacting proteins, immunoprecipitates were subjected to SDS-PAGE, and proteins were visualized by Colloidal Blue staining. (C) 293 cells were transfected with Flag-WDR5 or the control vector. Immunoprecipitations (IP) were performed with anti-Flag M2 antibodies. After washing, purified samples were subjected to SDS-PAGE followed by Western blot analysis (WB) using the antibodies indicated. (D) Recombinant CHD8 was incubated with recombinant GST or GST-WDR5 as indicated at the top. After washing, glutathione-agarose-purified samples were subjected to SDS-PAGE. The top of the gel was subjected to Western blot analysis using anti-CHD8 antibodies. The bottom of the gel was analyzed by Coomassie staining.

Techniques Used: Chromatography, Western Blot, Affinity Purification, Multiple Displacement Amplification, Immunoprecipitation, Incubation, SDS Page, Staining, Transfection, Plasmid Preparation, Purification, Recombinant

22) Product Images from "Regulation of atypical MAP kinases ERK3 and ERK4 by the phosphatase DUSP2"

Article Title: Regulation of atypical MAP kinases ERK3 and ERK4 by the phosphatase DUSP2

Journal: Scientific Reports

doi: 10.1038/srep43471

DUSP2 interacts with ERK3 and ERK4 in a KIM-dependent manner both in vitro and in vivo. Two micrograms of either ERK3 ( a ) or ERK4 ( b ) were incubated with 2 μg of GST, GST-mDUSP2, GST-mDUSP2KIM or GST-mDUSPC and glutathione agarose. Following GST pulldown, bound ERK3 or ERK4 was detected by western-blotting using an anti-ERK3 ( a ) or anti-ERK4 ( b ) antibody, respectively. GST and GST-fusions were visualized using an anti-GST antibody. ( c ) NCI-H1299 cells were transfected with either an empty expression vector or the plasmids encoding a myc-tagged catalytically inactive mutant of DUSP2 (DUSP2CS-Myc) or catalytically inactive DUSP2 in which the KIM motif was also mutated (DUSP2CSKIM-Myc) respectively. Twenty-four hours after transfection, cells were lysed and myc-tagged DUSP2 was immunoprecipitated from the lysate using an anti-Myc monoclonal antibody. Co-immunoprecipitated endogenous ERK3 was detected by Western-blotting using the anti-ERK3 (clone 4C11) antibody (upper panel) Immunoprecipitated DUSP2 was detected by Western-blotting using a sheep anti-DUSP2 antibody (second panel). The expression of endogenous ERK3 and overexpressed myc-DUSP2 in the lysates were verified by Western-blotting using a monoclonal anti-ERK3 (clone 4C11) antibody and polyclonal anti-DUSP2 antibody respectively (third and fourth panel). ( d ) HEK-293 cells were transfected with either empty expression vector or the plasmids DUSP2CS-HA or DUSP2CSKIM-HA respectively. Twenty-four hours after transfection cells were lysed and HA-tagged DUSP2 was immunoprecipitated from the lysate using an anti-HA monoclonal antibody. Co-immunoprecipitated endogenous ERK4 and ERK2 were detected by Western-blotting using the polyclonal anti-ERK4 antibody (upper panel) and polyclonal ERK2 antibody (second panel). Immunoprecipitated DUSP2 was detected by Western-blotting using a monoclonal anti-HA antibody (third panel). ( e ) Jurkat T-cells were stimulated with PMA and anti-CD3 antibody for 3 hours in presence of the proteosome inhibitor MG132. Endogenous ERK3 was immunoprecipitated from the cleared lysate using 2 ug goat polyclonal anti-ERK3 antibody (ERK3), a preimmune sheep IgG antibody (IgG) was used as a non-specific control. The immunoprecipitates were probed for ERK3 using a monoclonal anti-ERK3 antibody (clone 4C11, upper panel) and for DUSP2, (lower panel) using the anti-DUSP2 antibody. The presence of ERK3 and DUSP2 in the lysate was verified by western-blot of the lysate using the same antibodies. Unprocessed original scans of the blots are shown in Supplementary Fig. 1
Figure Legend Snippet: DUSP2 interacts with ERK3 and ERK4 in a KIM-dependent manner both in vitro and in vivo. Two micrograms of either ERK3 ( a ) or ERK4 ( b ) were incubated with 2 μg of GST, GST-mDUSP2, GST-mDUSP2KIM or GST-mDUSPC and glutathione agarose. Following GST pulldown, bound ERK3 or ERK4 was detected by western-blotting using an anti-ERK3 ( a ) or anti-ERK4 ( b ) antibody, respectively. GST and GST-fusions were visualized using an anti-GST antibody. ( c ) NCI-H1299 cells were transfected with either an empty expression vector or the plasmids encoding a myc-tagged catalytically inactive mutant of DUSP2 (DUSP2CS-Myc) or catalytically inactive DUSP2 in which the KIM motif was also mutated (DUSP2CSKIM-Myc) respectively. Twenty-four hours after transfection, cells were lysed and myc-tagged DUSP2 was immunoprecipitated from the lysate using an anti-Myc monoclonal antibody. Co-immunoprecipitated endogenous ERK3 was detected by Western-blotting using the anti-ERK3 (clone 4C11) antibody (upper panel) Immunoprecipitated DUSP2 was detected by Western-blotting using a sheep anti-DUSP2 antibody (second panel). The expression of endogenous ERK3 and overexpressed myc-DUSP2 in the lysates were verified by Western-blotting using a monoclonal anti-ERK3 (clone 4C11) antibody and polyclonal anti-DUSP2 antibody respectively (third and fourth panel). ( d ) HEK-293 cells were transfected with either empty expression vector or the plasmids DUSP2CS-HA or DUSP2CSKIM-HA respectively. Twenty-four hours after transfection cells were lysed and HA-tagged DUSP2 was immunoprecipitated from the lysate using an anti-HA monoclonal antibody. Co-immunoprecipitated endogenous ERK4 and ERK2 were detected by Western-blotting using the polyclonal anti-ERK4 antibody (upper panel) and polyclonal ERK2 antibody (second panel). Immunoprecipitated DUSP2 was detected by Western-blotting using a monoclonal anti-HA antibody (third panel). ( e ) Jurkat T-cells were stimulated with PMA and anti-CD3 antibody for 3 hours in presence of the proteosome inhibitor MG132. Endogenous ERK3 was immunoprecipitated from the cleared lysate using 2 ug goat polyclonal anti-ERK3 antibody (ERK3), a preimmune sheep IgG antibody (IgG) was used as a non-specific control. The immunoprecipitates were probed for ERK3 using a monoclonal anti-ERK3 antibody (clone 4C11, upper panel) and for DUSP2, (lower panel) using the anti-DUSP2 antibody. The presence of ERK3 and DUSP2 in the lysate was verified by western-blot of the lysate using the same antibodies. Unprocessed original scans of the blots are shown in Supplementary Fig. 1

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

DUSP2 dephosphorylates Serine 189 and 186 in the activation loop of ERK3 and ERK4 respectively in vivo . ( a ) HeLa cells were co-transfected with expression vectors encoding a kinase-dead mutant of ERK4 fused to green fluorescent protein (GFP-ERK4D168A) and either myc-tagged DUSP2, MKP-1, or MKP-3. After 24 h, whole cell extracts were prepared and GFP-ERK4D168A was immunoprecipitated using an anti-GFP antibody. The phosphorylation status of Serine 186 within the activation loop in immunoprecipitated ERK4 D168A was then analysed by Western blotting using an anti-phospho S186 antibody. The levels of GFP-ERK4D168A in immunoprecipitates and of the three MKPs in whole cell extracts were analysed by Western blotting using antibodies against ERK4 and myc, respectively. ( b ) HeLa cells were co-transfected with vectors encoding either myc-tagged ERK4 or ERK4D320N together with either HA-tagged DUSP2, DUSP2KIM, or DUSP2CS. After 24 h, whole cell extracts were prepared and either ERK4 or ERK4D320N were immunoprecipitated using an anti-myc antibody. The phosphorylation status of Serine 186 was analysed as described in A. Co-immunoprecipitated DUSP2 was visualized using an anti-HA antibody. The levels of overexpressed ERK4- and DUSP2 proteins were analysed by Western blotting of whole cell extracts using anti-myc and anti–HA antibodies, respectively. ( c ) NCI-H1299 cells were co-transfected with expression vectors encoding a Flag-tagged kinase-dead mutant of ERK3 (ERK3D171A) and either an empty expression vector or plasmids encoding wild-type DUSP2, a catalytically inactive DUSP2 mutant (DUSP2CS) or a kinase interaction motif-deficient mutant (DUSP2KIM) respectively. After 24 h, cell extracts were prepared and ERK3 was immunoprecipitated using M2-FLAG conjugated agarose. The Phosphorylation status of Serine 189 within the activation loop of immunoprecipitated ERK3 was analysed by Western-blotting using a specific anti-phospho Serine 189 ERK3 antibody. Levels of ERK3 protein in the input lysates and immunoprecipitates were analysed by Western-blotting using an anti-FLAG antibody. The expression of myc-tagged DUSP2 proteins in cell lysates and immunoprecipitates were analysed by Western-blotting using an anti-myc or anti-DUSP2 antibody respectively. All experiments were performed 3 times and representative images are shown. Unprocessed original scans of the blots are shown in Supplementary Fig. 1
Figure Legend Snippet: DUSP2 dephosphorylates Serine 189 and 186 in the activation loop of ERK3 and ERK4 respectively in vivo . ( a ) HeLa cells were co-transfected with expression vectors encoding a kinase-dead mutant of ERK4 fused to green fluorescent protein (GFP-ERK4D168A) and either myc-tagged DUSP2, MKP-1, or MKP-3. After 24 h, whole cell extracts were prepared and GFP-ERK4D168A was immunoprecipitated using an anti-GFP antibody. The phosphorylation status of Serine 186 within the activation loop in immunoprecipitated ERK4 D168A was then analysed by Western blotting using an anti-phospho S186 antibody. The levels of GFP-ERK4D168A in immunoprecipitates and of the three MKPs in whole cell extracts were analysed by Western blotting using antibodies against ERK4 and myc, respectively. ( b ) HeLa cells were co-transfected with vectors encoding either myc-tagged ERK4 or ERK4D320N together with either HA-tagged DUSP2, DUSP2KIM, or DUSP2CS. After 24 h, whole cell extracts were prepared and either ERK4 or ERK4D320N were immunoprecipitated using an anti-myc antibody. The phosphorylation status of Serine 186 was analysed as described in A. Co-immunoprecipitated DUSP2 was visualized using an anti-HA antibody. The levels of overexpressed ERK4- and DUSP2 proteins were analysed by Western blotting of whole cell extracts using anti-myc and anti–HA antibodies, respectively. ( c ) NCI-H1299 cells were co-transfected with expression vectors encoding a Flag-tagged kinase-dead mutant of ERK3 (ERK3D171A) and either an empty expression vector or plasmids encoding wild-type DUSP2, a catalytically inactive DUSP2 mutant (DUSP2CS) or a kinase interaction motif-deficient mutant (DUSP2KIM) respectively. After 24 h, cell extracts were prepared and ERK3 was immunoprecipitated using M2-FLAG conjugated agarose. The Phosphorylation status of Serine 189 within the activation loop of immunoprecipitated ERK3 was analysed by Western-blotting using a specific anti-phospho Serine 189 ERK3 antibody. Levels of ERK3 protein in the input lysates and immunoprecipitates were analysed by Western-blotting using an anti-FLAG antibody. The expression of myc-tagged DUSP2 proteins in cell lysates and immunoprecipitates were analysed by Western-blotting using an anti-myc or anti-DUSP2 antibody respectively. All experiments were performed 3 times and representative images are shown. Unprocessed original scans of the blots are shown in Supplementary Fig. 1

Techniques Used: Activation Assay, In Vivo, Transfection, Expressing, Mutagenesis, Immunoprecipitation, Western Blot, Plasmid Preparation

23) Product Images from "TOR-dependent reduction in the expression level of Rrn3p lowers the activity of the yeast RNA Pol I machinery, but does not account for the strong inhibition of rRNA production"

Article Title: TOR-dependent reduction in the expression level of Rrn3p lowers the activity of the yeast RNA Pol I machinery, but does not account for the strong inhibition of rRNA production

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkq264

Rrn3p levels are reduced upon TOR inactivation and proteasome-dependent degradation. ( A ) Rrn3p is degraded upon TOR inactivation and inhibition of translation. Yeast strain RRN3-Prot.A expressing a chromosomally Prot.A-tagged Rrn3p was grown in YPD at 30°C to mid-log phase (OD 600 ≈ 0.4; t = 0 min), before the cells were either treated with 200 ng/ml of rapamycin or with 100 µg/ml of cycloheximide, or starved in SDC-Leu (aa-depletion), respectively. At the time points indicated cells were collected and lysed. Same amounts of WCE (20 µg) were analysed by western blotting using antibodies directed against the Prot.A-tag of Rrn3p and the Pol I specific subunit A43, respectively. ( B ) Rrn3p-degradation depends on proteasome activity. The proteasome ts-mutant strain ( cim3-1, TOY 652 ) expressing a chromosomally TAP-tagged Rrn3p or the isogenic CIM3 WT strain (WT, TOY 651 ) were grown to mid-log phase in YPD at 24°C ( t = 0 min), before the cells were starved at 37°C in SDC-Leu medium (-Leu). At the time points indicated cells were collected and lysed. Same amounts of WCE (30 µg) were analysed by western blotting using antibodies directed against the TAP-tag of Rrn3p and the Pol I subunit A135, respectively. ( C ) Rrn3p is ubiquitylated. Yeast strain pNOP1-RRN3-Prot.A, expressing Prot.A-tagged Rrn3p from a plasmid was grown in YPD at 30°C to midlog phase, before half of the cells were treated with 200 ng/ml of rapamycin for 10 min. Cells of rapamycin treated and untreated cultures were collected and lysed. Same amounts of WCEs (6 mg) were incubated with either recombinant GST-Dsk2p, or recombinant GST immobilized on 50 µl of glutathione sepharose. After washing, proteins bound to the beads were eluted with SDS sample buffer. Same amounts (1%) (50 µg) of input (IN) and flow through (FT), 0.5% of the wash steps (washes) and 50% of the eluate (E) were analysed by western blotting using antibodies directed against the Prot.A-tag of Rrn3p.
Figure Legend Snippet: Rrn3p levels are reduced upon TOR inactivation and proteasome-dependent degradation. ( A ) Rrn3p is degraded upon TOR inactivation and inhibition of translation. Yeast strain RRN3-Prot.A expressing a chromosomally Prot.A-tagged Rrn3p was grown in YPD at 30°C to mid-log phase (OD 600 ≈ 0.4; t = 0 min), before the cells were either treated with 200 ng/ml of rapamycin or with 100 µg/ml of cycloheximide, or starved in SDC-Leu (aa-depletion), respectively. At the time points indicated cells were collected and lysed. Same amounts of WCE (20 µg) were analysed by western blotting using antibodies directed against the Prot.A-tag of Rrn3p and the Pol I specific subunit A43, respectively. ( B ) Rrn3p-degradation depends on proteasome activity. The proteasome ts-mutant strain ( cim3-1, TOY 652 ) expressing a chromosomally TAP-tagged Rrn3p or the isogenic CIM3 WT strain (WT, TOY 651 ) were grown to mid-log phase in YPD at 24°C ( t = 0 min), before the cells were starved at 37°C in SDC-Leu medium (-Leu). At the time points indicated cells were collected and lysed. Same amounts of WCE (30 µg) were analysed by western blotting using antibodies directed against the TAP-tag of Rrn3p and the Pol I subunit A135, respectively. ( C ) Rrn3p is ubiquitylated. Yeast strain pNOP1-RRN3-Prot.A, expressing Prot.A-tagged Rrn3p from a plasmid was grown in YPD at 30°C to midlog phase, before half of the cells were treated with 200 ng/ml of rapamycin for 10 min. Cells of rapamycin treated and untreated cultures were collected and lysed. Same amounts of WCEs (6 mg) were incubated with either recombinant GST-Dsk2p, or recombinant GST immobilized on 50 µl of glutathione sepharose. After washing, proteins bound to the beads were eluted with SDS sample buffer. Same amounts (1%) (50 µg) of input (IN) and flow through (FT), 0.5% of the wash steps (washes) and 50% of the eluate (E) were analysed by western blotting using antibodies directed against the Prot.A-tag of Rrn3p.

Techniques Used: Inhibition, Expressing, Western Blot, Activity Assay, Mutagenesis, Plasmid Preparation, Incubation, Recombinant, Flow Cytometry

Stabilization of cellular Rrn3p levels attenuates the reduction in initiation competent Pol I–Rrn3p complexes observed upon nutrient depletion. ( A ) Gelfiltration analysis. Yeast strains pNOP1-RRN3-Prot.A (WT) and pNOP1-RRN3-ΔN-Prot.A (ΔN) were grown in YPD at 30°C to mid-log phase. Cells were either starved for 2 h in SDC-Trp (-Trp) or further cultured in YPD and collected by centrifugation. After lysis, same amounts of WCE (900 µg) were separated on a Superose-6® column in a buffer containing 1.5 M potassium acetate. An amount of 250 µl of the collected 500 µl fractions were TCA precipitated and analysed by western blotting together with the ‘Load’ (30 µg). Antibodies used were directed against the Prot.A-tag of the Rrn3p versions and the Pol I subunit A135, respectively. The gel filtration fractions containing the initiation competent Pol I–Rrn3p complexes are labelled in red. ( B ) Co-immunoprecipitations. Yeast strains TOY 684 (WT) and TOY 685 (ΔN), both expressing chromosomally HA 3 -tagged Pol I subunit A43 and either full length or truncated Prot.A-tagged Rrn3p, were grown in YPD at 30°C to mid-log phase and half of the cells was crosslinked with 1% formaldehyde, harvested and lysed ( t = 0 min). The remainder of the cells was starved in SDC-Trp (-Trp) for 2 h and treated as described above ( t = 120 min). The HA 3 -tagged Pol I subunit A43 was immunoprecipitated (α-HA-IPs) from 250 µl of extracts (Inputs) with anti-HA antibody. Fifty percent of the α-HA-IPs as well as 1% of the inputs were analysed by western blotting using antibodies directed against the Prot.A-tag of the Rrn3p versions and the HA-tag of the Pol I subunit, respectively. As a control an identical co-immunoprecipitation experiment was performed using extracts from yeast strain pNOP1-RRN3-Prot.A and pNOP1-Rrn3-ΔN-Prot.A, which do not express the HA-tagged Pol I subunit A43 (ctr.). Western blot signal intensities were measured, and quantified using the LAS 3000 imaging system and the AIDA software. Rrn3p/A43 ratios were calculated, and the ratio of the 120 min samples was normalized to the ratio of the respective 0 min samples which was set to 100%. Numbers calculated are given below each lane. ( C ) Chromatin-IP (ChIP) experiments. Yeast strains pNOP1-RRN3-Prot.A (WT) and pNOP1-RRN3-ΔN-Prot.A (ΔN), both expressing either chromosomally HA 3 -tagged Pol I subunit A43 or the core-factor subunit Rrn6p, were grown in YPD at 30°C to mid-log phase and half of the cells was crosslinked with 1% formaldehyde, harvested, lysed and sonified. The remainder of the cells was starved in SDC-Trp for 2 h and treated as described above (-TRP). Rrn3p-Prot.A, the HA 3 -tagged Pol I subunit A43 or Rrn6p were immunoprecipitated from the chromatin extracts. After DNA isolation the relative amounts of specific DNA fragments co-purifying with the proteins were measured in triplicate real-time PCR reactions using primers specific for the rDNA promoter (P) and the 25S rRNA coding region (25S) as well as for the 5S rRNA gene (5S) which served as an internal control. Data were normalized to the promoter occupancy in growing wild-type cells and represent the mean of at least three independent ChIP experiments. ( D ) Reduction of 35S pre-rRNA synthesis is attenuated in the ΔN-mutant after TOR inactivation. (Upper panel) Yeast strains pNOP1-RRN3-Prot.A (WT) and pNOP1-RRN3-ΔN-Prot.A (ΔN) were cultured to mid-log phase ( t = 0 min), before the cells were treated with 200 ng/ml of rapamycin. At the time points indicated 5 ml of the cultures were pulse labelled for 15 min with 20 µCi of [ 5 , 6- 3 H] uracil, and total RNA was isolated. Equal amounts of total RNA were separated in a denaturing agarose gel and blotted onto a nylon membrane. 3 H-labelled RNAs were visualized and quantified using the BAS 1000 imaging system and the Image Gauge software. To determine the total RNA load per lane the membrane was hybridized with a 32 P-labelled oligonucleotide probe directed against the mature 25S rRNA (northern blot). Radioactive signals were visualized and quantified as described above. (Lower panel) The ratio of nascent 35S precursor rRNA to total 25S rRNA in the different rapamycin treated samples was determined and normalized to the 35S rRNA to 25S rRNA ratio of the untreated sample, which was arbitrarily set to 100.
Figure Legend Snippet: Stabilization of cellular Rrn3p levels attenuates the reduction in initiation competent Pol I–Rrn3p complexes observed upon nutrient depletion. ( A ) Gelfiltration analysis. Yeast strains pNOP1-RRN3-Prot.A (WT) and pNOP1-RRN3-ΔN-Prot.A (ΔN) were grown in YPD at 30°C to mid-log phase. Cells were either starved for 2 h in SDC-Trp (-Trp) or further cultured in YPD and collected by centrifugation. After lysis, same amounts of WCE (900 µg) were separated on a Superose-6® column in a buffer containing 1.5 M potassium acetate. An amount of 250 µl of the collected 500 µl fractions were TCA precipitated and analysed by western blotting together with the ‘Load’ (30 µg). Antibodies used were directed against the Prot.A-tag of the Rrn3p versions and the Pol I subunit A135, respectively. The gel filtration fractions containing the initiation competent Pol I–Rrn3p complexes are labelled in red. ( B ) Co-immunoprecipitations. Yeast strains TOY 684 (WT) and TOY 685 (ΔN), both expressing chromosomally HA 3 -tagged Pol I subunit A43 and either full length or truncated Prot.A-tagged Rrn3p, were grown in YPD at 30°C to mid-log phase and half of the cells was crosslinked with 1% formaldehyde, harvested and lysed ( t = 0 min). The remainder of the cells was starved in SDC-Trp (-Trp) for 2 h and treated as described above ( t = 120 min). The HA 3 -tagged Pol I subunit A43 was immunoprecipitated (α-HA-IPs) from 250 µl of extracts (Inputs) with anti-HA antibody. Fifty percent of the α-HA-IPs as well as 1% of the inputs were analysed by western blotting using antibodies directed against the Prot.A-tag of the Rrn3p versions and the HA-tag of the Pol I subunit, respectively. As a control an identical co-immunoprecipitation experiment was performed using extracts from yeast strain pNOP1-RRN3-Prot.A and pNOP1-Rrn3-ΔN-Prot.A, which do not express the HA-tagged Pol I subunit A43 (ctr.). Western blot signal intensities were measured, and quantified using the LAS 3000 imaging system and the AIDA software. Rrn3p/A43 ratios were calculated, and the ratio of the 120 min samples was normalized to the ratio of the respective 0 min samples which was set to 100%. Numbers calculated are given below each lane. ( C ) Chromatin-IP (ChIP) experiments. Yeast strains pNOP1-RRN3-Prot.A (WT) and pNOP1-RRN3-ΔN-Prot.A (ΔN), both expressing either chromosomally HA 3 -tagged Pol I subunit A43 or the core-factor subunit Rrn6p, were grown in YPD at 30°C to mid-log phase and half of the cells was crosslinked with 1% formaldehyde, harvested, lysed and sonified. The remainder of the cells was starved in SDC-Trp for 2 h and treated as described above (-TRP). Rrn3p-Prot.A, the HA 3 -tagged Pol I subunit A43 or Rrn6p were immunoprecipitated from the chromatin extracts. After DNA isolation the relative amounts of specific DNA fragments co-purifying with the proteins were measured in triplicate real-time PCR reactions using primers specific for the rDNA promoter (P) and the 25S rRNA coding region (25S) as well as for the 5S rRNA gene (5S) which served as an internal control. Data were normalized to the promoter occupancy in growing wild-type cells and represent the mean of at least three independent ChIP experiments. ( D ) Reduction of 35S pre-rRNA synthesis is attenuated in the ΔN-mutant after TOR inactivation. (Upper panel) Yeast strains pNOP1-RRN3-Prot.A (WT) and pNOP1-RRN3-ΔN-Prot.A (ΔN) were cultured to mid-log phase ( t = 0 min), before the cells were treated with 200 ng/ml of rapamycin. At the time points indicated 5 ml of the cultures were pulse labelled for 15 min with 20 µCi of [ 5 , 6- 3 H] uracil, and total RNA was isolated. Equal amounts of total RNA were separated in a denaturing agarose gel and blotted onto a nylon membrane. 3 H-labelled RNAs were visualized and quantified using the BAS 1000 imaging system and the Image Gauge software. To determine the total RNA load per lane the membrane was hybridized with a 32 P-labelled oligonucleotide probe directed against the mature 25S rRNA (northern blot). Radioactive signals were visualized and quantified as described above. (Lower panel) The ratio of nascent 35S precursor rRNA to total 25S rRNA in the different rapamycin treated samples was determined and normalized to the 35S rRNA to 25S rRNA ratio of the untreated sample, which was arbitrarily set to 100.

Techniques Used: Cell Culture, Centrifugation, Lysis, Western Blot, Filtration, Expressing, Immunoprecipitation, Imaging, Software, Chromatin Immunoprecipitation, DNA Extraction, Real-time Polymerase Chain Reaction, Mutagenesis, Isolation, Agarose Gel Electrophoresis, Northern Blot

24) Product Images from "Exportin 4 Interacts with Sox9 through the HMG Box and Inhibits the DNA Binding of Sox9"

Article Title: Exportin 4 Interacts with Sox9 through the HMG Box and Inhibits the DNA Binding of Sox9

Journal: PLoS ONE

doi: 10.1371/journal.pone.0025694

Identification of Exp4 as a major interaction partner of Sox9. (A) Silver staining of Sox9 binding proteins separated by NuPAGE. Nuclear extracts prepared from HeLa cells (HeLa NE) were incubated with (lanes 3, 4) or without FLAG-tagged Sox9 (FLAG-Sox9, lanes 1, 2). After recovery with anti-FLAG M2 antibody-conjugated agarose, the proteins were subjected to NuPAGE. The closed arrowhead indicates FLAG-Sox9, and the open arrowhead indicates the protein that was specifically recovered by FLAG-Sox9 (lane 4). (B) Nuclear extracts from U2OS cells were subjected to immunoprecipitation with anti-Sox9 antibody, and the precipitates were subjected to Western blotting analysis using anti-Exp4 antibody (right lane). Normal rabbit IgG was used as a control (middle lane). 1% of the nuclear extract was applied as a control (left lane). (C) The schematic depicts the truncated forms of Sox9 fused with GST (dark gray boxes). The numbers indicate the amino acid residues. The HMG box domain is shown as a light gray box (103–181 a.a.). (D) The upper panel shows Western blotting analysis of the protein samples co-precipitated with GST-fused truncated forms of Sox9 using an anti-Exp4 antibody. 5% of the nuclear extract was applied as a control (left lane). Numbers represent the corresponding GST-fused truncated Sox9 constructs shown in C. The lower panel shows CBB staining of NuPAGE for the GST fusion proteins used in this experiment. Numbers on the right represent the molecular weights of the marker proteins.
Figure Legend Snippet: Identification of Exp4 as a major interaction partner of Sox9. (A) Silver staining of Sox9 binding proteins separated by NuPAGE. Nuclear extracts prepared from HeLa cells (HeLa NE) were incubated with (lanes 3, 4) or without FLAG-tagged Sox9 (FLAG-Sox9, lanes 1, 2). After recovery with anti-FLAG M2 antibody-conjugated agarose, the proteins were subjected to NuPAGE. The closed arrowhead indicates FLAG-Sox9, and the open arrowhead indicates the protein that was specifically recovered by FLAG-Sox9 (lane 4). (B) Nuclear extracts from U2OS cells were subjected to immunoprecipitation with anti-Sox9 antibody, and the precipitates were subjected to Western blotting analysis using anti-Exp4 antibody (right lane). Normal rabbit IgG was used as a control (middle lane). 1% of the nuclear extract was applied as a control (left lane). (C) The schematic depicts the truncated forms of Sox9 fused with GST (dark gray boxes). The numbers indicate the amino acid residues. The HMG box domain is shown as a light gray box (103–181 a.a.). (D) The upper panel shows Western blotting analysis of the protein samples co-precipitated with GST-fused truncated forms of Sox9 using an anti-Exp4 antibody. 5% of the nuclear extract was applied as a control (left lane). Numbers represent the corresponding GST-fused truncated Sox9 constructs shown in C. The lower panel shows CBB staining of NuPAGE for the GST fusion proteins used in this experiment. Numbers on the right represent the molecular weights of the marker proteins.

Techniques Used: Silver Staining, Binding Assay, Incubation, Immunoprecipitation, Western Blot, Construct, Staining, Marker

Interaction of Exp4 with Sox family members. (A) Schematic representation of HA-tagged Sox proteins used in this study. The numbers indicate the amino acid residues. The HMG box domain is shown as a light gray box. The percentage of amino acid identity with the amino acid sequence of the HMG domain of Sox9 is given. (B) The panels show HA-affinity purification of proteins from extracts of HEK293 cells which were transiently transfected with FLAG-Exp4 and HA-Sox9, HA-Sox2, or HA-Sox11. Mock refers to the empty control plasmid. Starting materials (2% input) and bound fractions (IP, immunoprecipitation) were analyzed by NuPAGE and Western blotting. HA-tagged proteins are asterisked in the lower panels. The arrow indicates nonspecific bands. (C) The GST-fused HMG box domains of each Sox protein were separated by NuPAGE and stained with CBB (lower panel). The fusion proteins were incubated with recombinant Exp4 proteins. Proteins bound to glutathione-Sepharose were analyzed by Western blotting with anti-Exp4 antibody (upper panel). 20% input represents the control.
Figure Legend Snippet: Interaction of Exp4 with Sox family members. (A) Schematic representation of HA-tagged Sox proteins used in this study. The numbers indicate the amino acid residues. The HMG box domain is shown as a light gray box. The percentage of amino acid identity with the amino acid sequence of the HMG domain of Sox9 is given. (B) The panels show HA-affinity purification of proteins from extracts of HEK293 cells which were transiently transfected with FLAG-Exp4 and HA-Sox9, HA-Sox2, or HA-Sox11. Mock refers to the empty control plasmid. Starting materials (2% input) and bound fractions (IP, immunoprecipitation) were analyzed by NuPAGE and Western blotting. HA-tagged proteins are asterisked in the lower panels. The arrow indicates nonspecific bands. (C) The GST-fused HMG box domains of each Sox protein were separated by NuPAGE and stained with CBB (lower panel). The fusion proteins were incubated with recombinant Exp4 proteins. Proteins bound to glutathione-Sepharose were analyzed by Western blotting with anti-Exp4 antibody (upper panel). 20% input represents the control.

Techniques Used: Sequencing, Affinity Purification, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Staining, Incubation, Recombinant

25) Product Images from "Studies of Nematode TFIIE Function Reveal a Link between Ser-5 Phosphorylation of RNA Polymerase II and the Transition from Transcription Initiation to Elongation"

Article Title: Studies of Nematode TFIIE Function Reveal a Link between Ser-5 Phosphorylation of RNA Polymerase II and the Transition from Transcription Initiation to Elongation

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.21.1.1-15.2001

Identification of natural C. elegans TFIIE. (A) Coimmunoprecipitation of natural ceTFIIEα with ceTFIIEβ. To demonstrate association of natural ceTFIIEα with ceTFIIEβ, 80 μg of C. elegans embryonic nuclear extract was incubated with anti-ceTFIIEβ antibody–protein G-Sepharose, and natural ceTFIIEβ was precipitated. After SDS-PAGE on a 10% polyacrylamide gel, coprecipitated ceTFIIEα was detected with anti-ceTFIIEα rabbit antibody after Western blotting. Lane 1, 5% input of embryonic nuclear extract (4 μg); lane 2, recombinant ceTFIIEα (20 ng); lane 3, nuclear extract treated with preimmune serum (80 μg); lane 4, nuclear extract treated with anti-ceTFIIEβ serum (80 μg). An arrow indicates the position of ceTFIIEα (ceIIEα). (B) Coimmunoprecipitation of natural ceTFIIEβ with ceTFIIEα. The same strategy as detailed for panel A was employed to study the association of natural ceTFIIEβ with ceTFIIEα. Eighty micrograms of nuclear extract was incubated with anti-ceTFIIEα antibody–protein G-Sepharose, and natural ceTFIIEα was precipitated. Coprecipitated ceTFIIEβ was detected by anti-ceTFIIEβ rabbit antibody after Western blotting. Lane 1, 25% input of embryonic nuclear extract (20 μg); lane 2, nuclear extract treated with preimmune serum (80 μg); lanes 3 and 4, increasing amounts of nuclear extract treated with anti-ceTFIIEβ serum (40 and 80 μg, respectively); lane 5, recombinant ceTFIIEβ (200 ng). An arrow indicates the position of ceTFIIEβ (ceIIEβ). (C) Transcription complementation assay of ceTFIIE. ceTFIIE was depleted from nuclear extracts by treatment with anti-ceTFIIEβ antibody–protein G-Sepharose. In the same way, mock-depleted nuclear extracts were prepared by treatment with preimmune IgG–protein G-Sepharose. Complementation of natural ceTFIIE was studied by adding increasing amounts of purified recombinant ceTFIIE and carrying out primer extension reactions. Lane 1, ceTFIIE-depleted nuclear extracts (42 μg); lane 2, mock-depleted nuclear extracts (42 μg); lanes 3 to 5, ceTFIIE-depleted nuclear extracts (42 μg) with increasing amounts of recombinant ceTFIIE (5, 10, and 20 ng, respectively); lane 6, C. elegans nuclear extracts (42 μg). Arrows indicate the positions of the reverse transcript (92 nt) and the primer.
Figure Legend Snippet: Identification of natural C. elegans TFIIE. (A) Coimmunoprecipitation of natural ceTFIIEα with ceTFIIEβ. To demonstrate association of natural ceTFIIEα with ceTFIIEβ, 80 μg of C. elegans embryonic nuclear extract was incubated with anti-ceTFIIEβ antibody–protein G-Sepharose, and natural ceTFIIEβ was precipitated. After SDS-PAGE on a 10% polyacrylamide gel, coprecipitated ceTFIIEα was detected with anti-ceTFIIEα rabbit antibody after Western blotting. Lane 1, 5% input of embryonic nuclear extract (4 μg); lane 2, recombinant ceTFIIEα (20 ng); lane 3, nuclear extract treated with preimmune serum (80 μg); lane 4, nuclear extract treated with anti-ceTFIIEβ serum (80 μg). An arrow indicates the position of ceTFIIEα (ceIIEα). (B) Coimmunoprecipitation of natural ceTFIIEβ with ceTFIIEα. The same strategy as detailed for panel A was employed to study the association of natural ceTFIIEβ with ceTFIIEα. Eighty micrograms of nuclear extract was incubated with anti-ceTFIIEα antibody–protein G-Sepharose, and natural ceTFIIEα was precipitated. Coprecipitated ceTFIIEβ was detected by anti-ceTFIIEβ rabbit antibody after Western blotting. Lane 1, 25% input of embryonic nuclear extract (20 μg); lane 2, nuclear extract treated with preimmune serum (80 μg); lanes 3 and 4, increasing amounts of nuclear extract treated with anti-ceTFIIEβ serum (40 and 80 μg, respectively); lane 5, recombinant ceTFIIEβ (200 ng). An arrow indicates the position of ceTFIIEβ (ceIIEβ). (C) Transcription complementation assay of ceTFIIE. ceTFIIE was depleted from nuclear extracts by treatment with anti-ceTFIIEβ antibody–protein G-Sepharose. In the same way, mock-depleted nuclear extracts were prepared by treatment with preimmune IgG–protein G-Sepharose. Complementation of natural ceTFIIE was studied by adding increasing amounts of purified recombinant ceTFIIE and carrying out primer extension reactions. Lane 1, ceTFIIE-depleted nuclear extracts (42 μg); lane 2, mock-depleted nuclear extracts (42 μg); lanes 3 to 5, ceTFIIE-depleted nuclear extracts (42 μg) with increasing amounts of recombinant ceTFIIE (5, 10, and 20 ng, respectively); lane 6, C. elegans nuclear extracts (42 μg). Arrows indicate the positions of the reverse transcript (92 nt) and the primer.

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

26) Product Images from "The E3 Ubiquitin Ligase HAF1 Modulates Circadian Accumulation of EARLY FLOWERING3 to Control Heading Date in Rice under Long-Day Conditions [OPEN]"

Article Title: The E3 Ubiquitin Ligase HAF1 Modulates Circadian Accumulation of EARLY FLOWERING3 to Control Heading Date in Rice under Long-Day Conditions [OPEN]

Journal: The Plant Cell

doi: 10.1105/tpc.18.00653

HAF1 Interacts with OsELF3 in Vivo and in Vitro. (A) BiFC visualization of the interaction between HAF1 and OsELF3 in the nuclei of N. benthamiana epidermal cells. As negative controls, the substitution of leucine to serine (L558S) of OsELF3 or the substitution of glycine to cysteine (C620G) of HAF1 (HAF1) abolishes the interaction between these proteins. (B) BiFC showing the homodimer formation of OsELF3. Numbers on the right indicate the molecular masses of marker proteins (the unit is kilodaltons). OsELF3 (L558S) protein was used as a negative control. DAPI, fluorescence of 4′,6-diamino-2-phenylindole; DIC, differential interference contrast. (C) In vitro pull-down assay demonstrating the direct interaction between OsELF3 and HAF1. MBP-HAF1 was pulled down (PD) by GST-OsELF3 immobilized on glutathione-conjugated agarose beads and analyzed by immunoblotting (IB) using anti-MBP antibody. (D) In vitro pull-down assay showing that the C-terminal domain (amino acids 503–760) of OsELF3 is required for its homodimer formation.
Figure Legend Snippet: HAF1 Interacts with OsELF3 in Vivo and in Vitro. (A) BiFC visualization of the interaction between HAF1 and OsELF3 in the nuclei of N. benthamiana epidermal cells. As negative controls, the substitution of leucine to serine (L558S) of OsELF3 or the substitution of glycine to cysteine (C620G) of HAF1 (HAF1) abolishes the interaction between these proteins. (B) BiFC showing the homodimer formation of OsELF3. Numbers on the right indicate the molecular masses of marker proteins (the unit is kilodaltons). OsELF3 (L558S) protein was used as a negative control. DAPI, fluorescence of 4′,6-diamino-2-phenylindole; DIC, differential interference contrast. (C) In vitro pull-down assay demonstrating the direct interaction between OsELF3 and HAF1. MBP-HAF1 was pulled down (PD) by GST-OsELF3 immobilized on glutathione-conjugated agarose beads and analyzed by immunoblotting (IB) using anti-MBP antibody. (D) In vitro pull-down assay showing that the C-terminal domain (amino acids 503–760) of OsELF3 is required for its homodimer formation.

Techniques Used: In Vivo, In Vitro, Bimolecular Fluorescence Complementation Assay, Marker, Negative Control, Fluorescence, Pull Down Assay

27) Product Images from "Nuclear mRNA export requires specific FG nucleoporins for translocation through the nuclear pore complex"

Article Title: Nuclear mRNA export requires specific FG nucleoporins for translocation through the nuclear pore complex

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200704174

Mex67 binds the GLFG domain of Nup57. Bacterially expressed GST, GST-GLFG-NUP57, and GST-GLFG-NUP116 were each immobilized on glutathione agarose beads. Recombinant purified MBP-Mex67 was added, and the bound fraction was eluted. 10% of the input (MBP-Mex67) and the eluted fractions was resolved by SDS-PAGE and stained with Coomassie blue. Molecular mass (kilodaltons) markers are shown at the left ( M r ).
Figure Legend Snippet: Mex67 binds the GLFG domain of Nup57. Bacterially expressed GST, GST-GLFG-NUP57, and GST-GLFG-NUP116 were each immobilized on glutathione agarose beads. Recombinant purified MBP-Mex67 was added, and the bound fraction was eluted. 10% of the input (MBP-Mex67) and the eluted fractions was resolved by SDS-PAGE and stained with Coomassie blue. Molecular mass (kilodaltons) markers are shown at the left ( M r ).

Techniques Used: Recombinant, Purification, SDS Page, Staining

28) Product Images from "A safety study of newly generated anti-podoplanin-neutralizing antibody in cynomolgus monkey (Macaca fascicularis)"

Article Title: A safety study of newly generated anti-podoplanin-neutralizing antibody in cynomolgus monkey (Macaca fascicularis)

Journal: Oncotarget

doi: 10.18632/oncotarget.26055

Establishment of monkey podoplanin-neutralizing antibodies (A) Purification of a monkey podoplanin (mkyPDPN) immunogen to establish a hybridoma secreting anti-PDPN monoclonal antibodies (mAbs). A mkyPDPN cDNA region encoding amino acids 76–89 (226–267 bp) was tandemly connected 21 times. The cDNA fragment was inserted into a pGEX-6P-3 vector, and a glutathione S-transferase (GST)-tagged mkyPDPN peptide (76–89 aa) produced by BL21 (DE3) E. coli was purified using glutathione sepharose. BALB/c mice were injected with the GST-tagged peptide, after which their splenocytes were fused with mouse myeloma P3U1 cells using polyethylene glycol. Hybridoma screening and antibody purification from ascites were performed. (B) CHO cells transfected with an empty vector (mock), wild-type monkey podoplanin (mkyPDPN-WT), wild-type human podoplanin (hPDPN-WT) were treated with PBS (closed areas) or antibodies (open areas), including anti-PDPN mAb D2-40, 1F6, 2F7, or 3F4 to measure PDPN expression levels. (C) GST-tagged recombinant mkyPDPN protein (WT) and its point mutants were expressed in E. coli . Cell lysates were electrophoresed and immunoblotted with antibodies to PDPN (1F6, 2F7, 3F4) or GST. The PLAG4 domain is indicated by red letters. (D) CHO/mock, CHO/mkyPDPN-WT, or CHO/hPDPN-WT cells were incubated with 100 μg/mL of control IgG1 or anti-PDPN antibodies 1F6, 2F7, or 3F4, followed by incubation with 1 μg/mL of hCLEC-2-(His) 10 (open areas: control IgG-treated samples; green areas: anti-PDPN mAb-treated samples). After washing, cells were further incubated with Alexa Flour 488-conjugated anti-penta-His second antibody. CLEC-2 binding was measured by flow cytometry. Gray areas indicate the fluorescence intensity of samples not treated with CLEC-2. (E) CHO/mkyPDPN-WT cells were incubated with 10 μg/mL of control IgG1, 1F6, 2F7, or 3F4 mAbs followed by incubation with mouse platelet-rich plasma (PRP). The aggregation rate was estimated using an aggregometer. (F) PLAG3 domain-deleted mkyPDPN mutant cells were incubated with 10 μg/mL of control IgG1, 1F6, 2F7, or 3F4 mAbs, followed by incubation with mouse PRP. The aggregation rate was estimated using an aggregometer.
Figure Legend Snippet: Establishment of monkey podoplanin-neutralizing antibodies (A) Purification of a monkey podoplanin (mkyPDPN) immunogen to establish a hybridoma secreting anti-PDPN monoclonal antibodies (mAbs). A mkyPDPN cDNA region encoding amino acids 76–89 (226–267 bp) was tandemly connected 21 times. The cDNA fragment was inserted into a pGEX-6P-3 vector, and a glutathione S-transferase (GST)-tagged mkyPDPN peptide (76–89 aa) produced by BL21 (DE3) E. coli was purified using glutathione sepharose. BALB/c mice were injected with the GST-tagged peptide, after which their splenocytes were fused with mouse myeloma P3U1 cells using polyethylene glycol. Hybridoma screening and antibody purification from ascites were performed. (B) CHO cells transfected with an empty vector (mock), wild-type monkey podoplanin (mkyPDPN-WT), wild-type human podoplanin (hPDPN-WT) were treated with PBS (closed areas) or antibodies (open areas), including anti-PDPN mAb D2-40, 1F6, 2F7, or 3F4 to measure PDPN expression levels. (C) GST-tagged recombinant mkyPDPN protein (WT) and its point mutants were expressed in E. coli . Cell lysates were electrophoresed and immunoblotted with antibodies to PDPN (1F6, 2F7, 3F4) or GST. The PLAG4 domain is indicated by red letters. (D) CHO/mock, CHO/mkyPDPN-WT, or CHO/hPDPN-WT cells were incubated with 100 μg/mL of control IgG1 or anti-PDPN antibodies 1F6, 2F7, or 3F4, followed by incubation with 1 μg/mL of hCLEC-2-(His) 10 (open areas: control IgG-treated samples; green areas: anti-PDPN mAb-treated samples). After washing, cells were further incubated with Alexa Flour 488-conjugated anti-penta-His second antibody. CLEC-2 binding was measured by flow cytometry. Gray areas indicate the fluorescence intensity of samples not treated with CLEC-2. (E) CHO/mkyPDPN-WT cells were incubated with 10 μg/mL of control IgG1, 1F6, 2F7, or 3F4 mAbs followed by incubation with mouse platelet-rich plasma (PRP). The aggregation rate was estimated using an aggregometer. (F) PLAG3 domain-deleted mkyPDPN mutant cells were incubated with 10 μg/mL of control IgG1, 1F6, 2F7, or 3F4 mAbs, followed by incubation with mouse PRP. The aggregation rate was estimated using an aggregometer.

Techniques Used: Purification, Plasmid Preparation, Produced, Mouse Assay, Injection, Antibody Purification, Transfection, Expressing, Recombinant, Incubation, Binding Assay, Flow Cytometry, Cytometry, Fluorescence, Mutagenesis

29) Product Images from "BRCA1 interacts with components of the histone deacetylase complex"

Article Title: BRCA1 interacts with components of the histone deacetylase complex

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

doi:

The BRCT domain binds to Rb-binding proteins in vitro . ( A ) Schematic representation of BRCA1 gene. The region used as a probe in the library screen is indicated. ( B ) GST pull-down assays demonstrating that BRCT domain interacts with partial polypeptide of RbAp46 and full-length RbAp46 and RbAp48 proteins. In vitro -translated, 35 S-labeled BRCT (10 μl) was incubated with 20 μl of glutathione-Sepharose beads and an equal amount of GST or GST-fusion proteins, as indicated. After extensive washing, bound proteins were eluted, resolved on SDS/10% PAGE, and visualized by using autoradiography. A portion of the in vitro -translated, 35 S-labeled BRCT, corresponding to ≈20% of the labeled protein in the binding reaction, was loaded as “Input”. ( C ) Schematic representation of the BRCT repeats. Mutations analyzed for in vitro binding are indicated. ( D ) Mutations in BRCT domain interfere with interaction between RbAp46 and BRCT. GST pull-down assays were performed as in A with the wild type or mutation-containing BRCT polypeptides as indicated. Approximately 5% of each in vitro -translated protein in the binding reactions were loaded as Input.
Figure Legend Snippet: The BRCT domain binds to Rb-binding proteins in vitro . ( A ) Schematic representation of BRCA1 gene. The region used as a probe in the library screen is indicated. ( B ) GST pull-down assays demonstrating that BRCT domain interacts with partial polypeptide of RbAp46 and full-length RbAp46 and RbAp48 proteins. In vitro -translated, 35 S-labeled BRCT (10 μl) was incubated with 20 μl of glutathione-Sepharose beads and an equal amount of GST or GST-fusion proteins, as indicated. After extensive washing, bound proteins were eluted, resolved on SDS/10% PAGE, and visualized by using autoradiography. A portion of the in vitro -translated, 35 S-labeled BRCT, corresponding to ≈20% of the labeled protein in the binding reaction, was loaded as “Input”. ( C ) Schematic representation of the BRCT repeats. Mutations analyzed for in vitro binding are indicated. ( D ) Mutations in BRCT domain interfere with interaction between RbAp46 and BRCT. GST pull-down assays were performed as in A with the wild type or mutation-containing BRCT polypeptides as indicated. Approximately 5% of each in vitro -translated protein in the binding reactions were loaded as Input.

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

Rb interacts with the BRCT domain. ( A ) GST pull-down experiments show that Rb interacts with BRCT domain in the presence or absence of partial RbAp46 polypeptide. In vitro -translated, 35 S-labeled BRCT (12.5 μl) and partial RbAp46 polypeptides were incubated individually or in combination with 20 μl of glutathione-Sepharose beads with equal amount of GST-Rb or GST-Rb pocket mutation fusion proteins as indicated. GST-coated beads were incubated with each polypeptide separately. After extensive washing, bound proteins were eluted, resolved on SDS/10% PAGE and visualized by using autoradiography. Approximately 20% of labeled protein in the binding reaction were loaded as Input. ( B ) Colocalization of BRCA1 and Rb. HeLa cells were prepared as described in Material and Methods , stained with a mouse mAb against Rb, IF8, (green in a ), a rabbit polyclonal antibody against BRCA1, I-20, (red in b ). The regions of overlap between red and green signals appear as yellow ( c ), indicating colocalization of BRCA1 and Rb. The nucleus of each cells are shown by DAPI staining ( d ).
Figure Legend Snippet: Rb interacts with the BRCT domain. ( A ) GST pull-down experiments show that Rb interacts with BRCT domain in the presence or absence of partial RbAp46 polypeptide. In vitro -translated, 35 S-labeled BRCT (12.5 μl) and partial RbAp46 polypeptides were incubated individually or in combination with 20 μl of glutathione-Sepharose beads with equal amount of GST-Rb or GST-Rb pocket mutation fusion proteins as indicated. GST-coated beads were incubated with each polypeptide separately. After extensive washing, bound proteins were eluted, resolved on SDS/10% PAGE and visualized by using autoradiography. Approximately 20% of labeled protein in the binding reaction were loaded as Input. ( B ) Colocalization of BRCA1 and Rb. HeLa cells were prepared as described in Material and Methods , stained with a mouse mAb against Rb, IF8, (green in a ), a rabbit polyclonal antibody against BRCA1, I-20, (red in b ). The regions of overlap between red and green signals appear as yellow ( c ), indicating colocalization of BRCA1 and Rb. The nucleus of each cells are shown by DAPI staining ( d ).

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

The BRCT domain associates with HDAC1 and HDAC2. Western blot analysis of GST pull-down assays using antibodies against HDAC1 and HDAC2. HeLa whole-cell lysates (300 μg) were incubated with 20 μl of glutathione-Sepharose beads covered with equal amounts of GST, GST-BRCT (amino acids 1,536–1,863), or GST-NH 2 -BRCA1 (amino acids 1–304) fusion proteins. Bound proteins were resolved on SDS/10% PAGE, transferred to nitrocellulose membranes, and blotted with HDAC1 or HDAC2 Abs.
Figure Legend Snippet: The BRCT domain associates with HDAC1 and HDAC2. Western blot analysis of GST pull-down assays using antibodies against HDAC1 and HDAC2. HeLa whole-cell lysates (300 μg) were incubated with 20 μl of glutathione-Sepharose beads covered with equal amounts of GST, GST-BRCT (amino acids 1,536–1,863), or GST-NH 2 -BRCA1 (amino acids 1–304) fusion proteins. Bound proteins were resolved on SDS/10% PAGE, transferred to nitrocellulose membranes, and blotted with HDAC1 or HDAC2 Abs.

Techniques Used: Western Blot, Incubation, Polyacrylamide Gel Electrophoresis

30) Product Images from "The RNA polymerase II C-terminal domain promotes splicing activation through recruitment of a U2AF65-Prp19 complex"

Article Title: The RNA polymerase II C-terminal domain promotes splicing activation through recruitment of a U2AF65-Prp19 complex

Journal: Genes & Development

doi: 10.1101/gad.2038011

U2AF and PRP19C are required for CTD-dependent splicing activity. ( A ) NF20–40 was depleted of U2AF65 at 1 M KCl using Poly-U sepharose beads, followed by immunoblotting against U2AF65 and CDC5L. Band intensities quantified using Li-cor software are indicated below . ( B , lanes 2 and 3 ) Mock and depleted extracts from A were used in the CTD-dependent splicing assay. One-hundred nanomolar U2AF65 and U2AF produced in baculovirus-infected insect cells were tested for their ability to complement the depleted NF20–40 (lanes 4 , 5 ) or activate splicing when added alone (lanes 6 , 7 ). Relative intensities of spliced product were quantified using ImageQuant software and are indicated below . ( C ) PRP19C was depleted from an active Mono Q fraction at 500 mM NaCl using an anti-CDC5L antibody or was mock-depleted using an anti-Flag antibody. Mock and depleted samples were immunoblotted for U2AF65 and CDC5L. Relative CDC5L levels were normalized to U2AF65 and are indicated below . ( D ) CTD-dependent splicing assays were performed using the mock- and PRP19C-depleted samples from C . Relative amounts of spliced product are indicated below .
Figure Legend Snippet: U2AF and PRP19C are required for CTD-dependent splicing activity. ( A ) NF20–40 was depleted of U2AF65 at 1 M KCl using Poly-U sepharose beads, followed by immunoblotting against U2AF65 and CDC5L. Band intensities quantified using Li-cor software are indicated below . ( B , lanes 2 and 3 ) Mock and depleted extracts from A were used in the CTD-dependent splicing assay. One-hundred nanomolar U2AF65 and U2AF produced in baculovirus-infected insect cells were tested for their ability to complement the depleted NF20–40 (lanes 4 , 5 ) or activate splicing when added alone (lanes 6 , 7 ). Relative intensities of spliced product were quantified using ImageQuant software and are indicated below . ( C ) PRP19C was depleted from an active Mono Q fraction at 500 mM NaCl using an anti-CDC5L antibody or was mock-depleted using an anti-Flag antibody. Mock and depleted samples were immunoblotted for U2AF65 and CDC5L. Relative CDC5L levels were normalized to U2AF65 and are indicated below . ( D ) CTD-dependent splicing assays were performed using the mock- and PRP19C-depleted samples from C . Relative amounts of spliced product are indicated below .

Techniques Used: Activity Assay, Software, Splicing Assay, Produced, Infection

U2AF65 interacts with PRP19C in an RNA-independent manner. ( A , left ) Schematic of U2AF65 and deletion constructs used for GST pull-downs. ( Right ) These constructs were expressed in E. coli and then purified by GSH sepharose beads. The beads were run on SDS-PAGE and Coomassie-stained. ( B ) GST pull-down was performed using GST-tagged U2AF65, U2AF65ΔRS, or U2AF65ΔUHM. After a 3-h incubation at 22°C with the indicated extract, beads were washed and then eluted with 15 mM glutathione. Eluted proteins were immunoblotted with an anti-Prp19 antibody. ( C ) A vector expressing Flag-tagged SPF27 was transfected into 293T cells, cells were harvested and used to make NE, and PRP19C was purified using anti-Flag agarose and washed with buffer D containing the indicated salt concentration. In parallel, beads were incubated with NE from nontransfected cells amM KCl. Beads were eluted with Flag peptide, separated by SDS-PAGE, and Coomassie stained ( left ) or immunoblotted with the indicated antibody ( right ). ( D ) Co-IPs were carried out in NF20–40 using anti-Prp19 antibodies or anti-GST antibodies (mock). Beads were washed with buffer D and then boiled and immunoblotted for U2AF65. ( E ) Co-IP was performed using anti-CDC5L antibodies or anti-GST antibodies as above, except NF20–40 was mock- or RNase A-treated beforehand for 30 min at 37°C. The effectiveness of RNase A digestion was confirmed by agarose gel electrophoresis followed by ethidium staining (not shown).
Figure Legend Snippet: U2AF65 interacts with PRP19C in an RNA-independent manner. ( A , left ) Schematic of U2AF65 and deletion constructs used for GST pull-downs. ( Right ) These constructs were expressed in E. coli and then purified by GSH sepharose beads. The beads were run on SDS-PAGE and Coomassie-stained. ( B ) GST pull-down was performed using GST-tagged U2AF65, U2AF65ΔRS, or U2AF65ΔUHM. After a 3-h incubation at 22°C with the indicated extract, beads were washed and then eluted with 15 mM glutathione. Eluted proteins were immunoblotted with an anti-Prp19 antibody. ( C ) A vector expressing Flag-tagged SPF27 was transfected into 293T cells, cells were harvested and used to make NE, and PRP19C was purified using anti-Flag agarose and washed with buffer D containing the indicated salt concentration. In parallel, beads were incubated with NE from nontransfected cells amM KCl. Beads were eluted with Flag peptide, separated by SDS-PAGE, and Coomassie stained ( left ) or immunoblotted with the indicated antibody ( right ). ( D ) Co-IPs were carried out in NF20–40 using anti-Prp19 antibodies or anti-GST antibodies (mock). Beads were washed with buffer D and then boiled and immunoblotted for U2AF65. ( E ) Co-IP was performed using anti-CDC5L antibodies or anti-GST antibodies as above, except NF20–40 was mock- or RNase A-treated beforehand for 30 min at 37°C. The effectiveness of RNase A digestion was confirmed by agarose gel electrophoresis followed by ethidium staining (not shown).

Techniques Used: Construct, Purification, SDS Page, Staining, Incubation, Plasmid Preparation, Expressing, Transfection, Concentration Assay, Co-Immunoprecipitation Assay, Agarose Gel Electrophoresis

31) Product Images from "Histone phosphorylation by TRPM6’s cleaved kinase attenuates adjacent arginine methylation to regulate gene expression"

Article Title: Histone phosphorylation by TRPM6’s cleaved kinase attenuates adjacent arginine methylation to regulate gene expression

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

doi: 10.1073/pnas.1708427114

Santa Cruz sc-365536 mouse monoclonal antibody (sc) recognizes C-terminal TRPM6 fragments larger than ∼70 kDa. C-terminally HA-tagged TRPM6 stably expressed in 293T cells, immunoprecipitated with anti-HA agarose, and probed on Western blot with anti–HA-peroxidase or sc-365536. Thus, the sc-365536 epitope is located N-terminal to amino acid ∼1400.
Figure Legend Snippet: Santa Cruz sc-365536 mouse monoclonal antibody (sc) recognizes C-terminal TRPM6 fragments larger than ∼70 kDa. C-terminally HA-tagged TRPM6 stably expressed in 293T cells, immunoprecipitated with anti-HA agarose, and probed on Western blot with anti–HA-peroxidase or sc-365536. Thus, the sc-365536 epitope is located N-terminal to amino acid ∼1400.

Techniques Used: Stable Transfection, Immunoprecipitation, Western Blot

M6CK binds PRMT5, WDR77, and ICLN subunits of the protein methylase complex. ( A ) Endogenous TRPM6 binds endogenous PRMT5 and associated proteins. Extracts of 293T cells with endogenous HA-tagged TRPM6 (TRPM6 HA ) immunoprecipitated with HA-agarose and probed on WB with antibodies recognizing proteins of interest. Parental 293T cells (WT) served as negative controls. ( B ) Extracts of 293T cells stably expressing FLAG-tagged full-length TRPM6 or M6CK (first amino acid is residue 1365 of full-length TRPM6) immunoprecipitated with FLAG-agarose and probed on Western blot with antibodies recognizing proteins of interest. Parental 293T cells (WT) served as negative controls.
Figure Legend Snippet: M6CK binds PRMT5, WDR77, and ICLN subunits of the protein methylase complex. ( A ) Endogenous TRPM6 binds endogenous PRMT5 and associated proteins. Extracts of 293T cells with endogenous HA-tagged TRPM6 (TRPM6 HA ) immunoprecipitated with HA-agarose and probed on WB with antibodies recognizing proteins of interest. Parental 293T cells (WT) served as negative controls. ( B ) Extracts of 293T cells stably expressing FLAG-tagged full-length TRPM6 or M6CK (first amino acid is residue 1365 of full-length TRPM6) immunoprecipitated with FLAG-agarose and probed on Western blot with antibodies recognizing proteins of interest. Parental 293T cells (WT) served as negative controls.

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

32) Product Images from "Cyclophilins Facilitate Dissociation of the Human Papillomavirus Type 16 Capsid Protein L1 from the L2/DNA Complex following Virus Entry"

Article Title: Cyclophilins Facilitate Dissociation of the Human Papillomavirus Type 16 Capsid Protein L1 from the L2/DNA Complex following Virus Entry

Journal: Journal of Virology

doi: 10.1128/JVI.00980-12

In vitro dissociation of HPV11 L1 from complexes of L1 and GST-L2 by CyPs. (A) Sequence comparison of putative CyP binding sites. (B) Complexes of L1 and GST-L2 bound to glutathione Sepharose beads were incubated with CyPA, CyPB, or buffer control at
Figure Legend Snippet: In vitro dissociation of HPV11 L1 from complexes of L1 and GST-L2 by CyPs. (A) Sequence comparison of putative CyP binding sites. (B) Complexes of L1 and GST-L2 bound to glutathione Sepharose beads were incubated with CyPA, CyPB, or buffer control at

Techniques Used: In Vitro, Sequencing, Binding Assay, Incubation

33) Product Images from "Calcineurin-mediated Dephosphorylation of Synaptotagmin VI Is Necessary for Acrosomal Exocytosis *"

Article Title: Calcineurin-mediated Dephosphorylation of Synaptotagmin VI Is Necessary for Acrosomal Exocytosis *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.095752

Synaptotagmin is a substrate for calcineurin. A–C , GST-C2B domains immobilized in glutathione-Sepharose beads were incubated for 40 min at 37 °C with PKCβII under activating conditions in the presence of [γ- 32 P]ATP. The
Figure Legend Snippet: Synaptotagmin is a substrate for calcineurin. A–C , GST-C2B domains immobilized in glutathione-Sepharose beads were incubated for 40 min at 37 °C with PKCβII under activating conditions in the presence of [γ- 32 P]ATP. The

Techniques Used: Incubation

34) Product Images from "A Docking Site in MKK4 Mediates High Affinity Binding to JNK MAPKs and Competes with Similar Docking Sites in JNK Substrates *"

Article Title: A Docking Site in MKK4 Mediates High Affinity Binding to JNK MAPKs and Competes with Similar Docking Sites in JNK Substrates *

Journal: The Journal of biological chemistry

doi: 10.1074/jbc.M304229200

The MKK4 D-site is required for JNK binding A , 35 S-labeled MKK4 derivatives were tested for binding to GST-JNK3. B , 35 S-radiolabeled full-length MKK4 protein and N-terminal truncations thereof were prepared by in vitro translation and partially purified by ammonium sulfate precipitation, and portions (10% of the amount added in the binding reactions) were resolved on a 10% SDS-polyacrylamide gel ( lane 1 ). Samples (~1 pmol) of the same proteins were incubated with > 40 μ g of GST ( lane 2 ) or with 10 or 40 μ g of GST-JNK3 ( lanes 3 and 4 , respectively) bound to glutathione-Sepharose beads, and the resulting bead-bound protein complexes were isolated by sedimentation and resolved by 10% SDS-PAGE on the same gel. The gel was analyzed by staining with Coomassie Blue for visualization of the bound GST fusion protein (a representative example is shown in the lowest panel ) and by autoradiography for visualization of the bound radiolabeled protein ( upper three panels ).
Figure Legend Snippet: The MKK4 D-site is required for JNK binding A , 35 S-labeled MKK4 derivatives were tested for binding to GST-JNK3. B , 35 S-radiolabeled full-length MKK4 protein and N-terminal truncations thereof were prepared by in vitro translation and partially purified by ammonium sulfate precipitation, and portions (10% of the amount added in the binding reactions) were resolved on a 10% SDS-polyacrylamide gel ( lane 1 ). Samples (~1 pmol) of the same proteins were incubated with > 40 μ g of GST ( lane 2 ) or with 10 or 40 μ g of GST-JNK3 ( lanes 3 and 4 , respectively) bound to glutathione-Sepharose beads, and the resulting bead-bound protein complexes were isolated by sedimentation and resolved by 10% SDS-PAGE on the same gel. The gel was analyzed by staining with Coomassie Blue for visualization of the bound GST fusion protein (a representative example is shown in the lowest panel ) and by autoradiography for visualization of the bound radiolabeled protein ( upper three panels ).

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

35) Product Images from "A Bacillus anthracis S-Layer Homology Protein That Binds Heme and Mediates Heme Delivery to IsdC ▿"

Article Title: A Bacillus anthracis S-Layer Homology Protein That Binds Heme and Mediates Heme Delivery to IsdC ▿

Journal: Journal of Bacteriology

doi: 10.1128/JB.00054-10

Heme transfer to apo-IsdC. (A) Outline of the procedure to monitor heme transfer from holo-BslK N to buffer (line 1), BSA (line 2), apo-IsdX1 (line 3), or apo-IsdC (line 4). (B) GST-(holo)BslK N , coupled to glutathione-Sepharose, was incubated with equimolar
Figure Legend Snippet: Heme transfer to apo-IsdC. (A) Outline of the procedure to monitor heme transfer from holo-BslK N to buffer (line 1), BSA (line 2), apo-IsdX1 (line 3), or apo-IsdC (line 4). (B) GST-(holo)BslK N , coupled to glutathione-Sepharose, was incubated with equimolar

Techniques Used: Incubation

36) Product Images from "EBAG9 Adds a New Layer of Control on Large Dense-Core Vesicle Exocytosis via Interaction with Snapin D⃞"

Article Title: EBAG9 Adds a New Layer of Control on Large Dense-Core Vesicle Exocytosis via Interaction with Snapin D⃞

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E04-09-0817

EBAG9 interacts with Snapin in vitro and in vivo. (A) GST-Snapin, GST-SNAP25, GST-VAMP2, or GST (3 μg each) immobilized on glutathione-Sepharose beads were incubated with in vitro-translated 35 S-labeled EBAG9. Bound proteins were analyzed by 12.5%
Figure Legend Snippet: EBAG9 interacts with Snapin in vitro and in vivo. (A) GST-Snapin, GST-SNAP25, GST-VAMP2, or GST (3 μg each) immobilized on glutathione-Sepharose beads were incubated with in vitro-translated 35 S-labeled EBAG9. Bound proteins were analyzed by 12.5%

Techniques Used: In Vitro, In Vivo, Incubation, Labeling

37) Product Images from "Yeast Gga Coat Proteins Function with Clathrin in Golgi to Endosome Transport"

Article Title: Yeast Gga Coat Proteins Function with Clathrin in Golgi to Endosome Transport

Journal: Molecular Biology of the Cell

doi:

Interaction of Gga proteins and clathrin in vitro. The C-terminal half of Gga1p (aa 282–585) fused to GST (GST-Gga1C) or GST was bound to glutathione-Sepharose beads and incubated with extract from TVY614. Proteins bound to GST beads (lane 2) or GST-Gga1C beads (lane 3) were analyzed by SDS-PAGE followed by immunoblotting (A) or Coomassie blue staining (B) and compared with extract from TVY614 (input, lane 1). Inputs correspond to 100% (A) or 10% (B) of the amount of extract incubated with beads. (C) Extract from cells expressing Gga2p-3HA (GPY2373; lane 1) was subjected to nondenaturing immunoprecipitation with the use of anti-HA antibodies (IP, lane 2), followed by SDS-PAGE and immunoblotting. The input corresponds to 2% of the amount of extract subjected to immunoprecipitation.
Figure Legend Snippet: Interaction of Gga proteins and clathrin in vitro. The C-terminal half of Gga1p (aa 282–585) fused to GST (GST-Gga1C) or GST was bound to glutathione-Sepharose beads and incubated with extract from TVY614. Proteins bound to GST beads (lane 2) or GST-Gga1C beads (lane 3) were analyzed by SDS-PAGE followed by immunoblotting (A) or Coomassie blue staining (B) and compared with extract from TVY614 (input, lane 1). Inputs correspond to 100% (A) or 10% (B) of the amount of extract incubated with beads. (C) Extract from cells expressing Gga2p-3HA (GPY2373; lane 1) was subjected to nondenaturing immunoprecipitation with the use of anti-HA antibodies (IP, lane 2), followed by SDS-PAGE and immunoblotting. The input corresponds to 2% of the amount of extract subjected to immunoprecipitation.

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

38) Product Images from "Ribosomal Protein S3 Negatively Regulates Unwinding Activity of RecQ-like Helicase 4 through Their Physical Interaction *"

Article Title: Ribosomal Protein S3 Negatively Regulates Unwinding Activity of RecQ-like Helicase 4 through Their Physical Interaction *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M116.764324

RECQL4 physically interacts with RPS3. A , nuclear lysates of 293 cell lines stably expressing RECQL4-V5-his or control plasmid are used for V5 pulldown followed by anti-RPS3, anti-V5 and anti-Lamin B immunoblotting. B , Coomassie Blue-stained SDS-PAGE depicting final purified protein fraction of GST-FL-RPS3 after Biorex TM 70 and glutathione-Sepharose purification steps. C , GST pulldown assay. Anti-RECQL4 and anti-GST blots show input and pulldown fractions of GST pulldown assay carried out by incubating RECQL4-his with GST-RPS3 or GST only proteins. D , confocal images of representative U2OS cells immunostained by anti-RECQL4, anti-RPS3 antibodies, and DAPI. The plot shows nuclear RPS3 (%) and nuclear colocalization of RPS3-RECQL4 (%) quantified from three-dimensional reconstructions of Z section confocal images, using Imaris software. The data are shown as the means ± S.D. of triplicate experiments.
Figure Legend Snippet: RECQL4 physically interacts with RPS3. A , nuclear lysates of 293 cell lines stably expressing RECQL4-V5-his or control plasmid are used for V5 pulldown followed by anti-RPS3, anti-V5 and anti-Lamin B immunoblotting. B , Coomassie Blue-stained SDS-PAGE depicting final purified protein fraction of GST-FL-RPS3 after Biorex TM 70 and glutathione-Sepharose purification steps. C , GST pulldown assay. Anti-RECQL4 and anti-GST blots show input and pulldown fractions of GST pulldown assay carried out by incubating RECQL4-his with GST-RPS3 or GST only proteins. D , confocal images of representative U2OS cells immunostained by anti-RECQL4, anti-RPS3 antibodies, and DAPI. The plot shows nuclear RPS3 (%) and nuclear colocalization of RPS3-RECQL4 (%) quantified from three-dimensional reconstructions of Z section confocal images, using Imaris software. The data are shown as the means ± S.D. of triplicate experiments.

Techniques Used: Stable Transfection, Expressing, Plasmid Preparation, Staining, SDS Page, Purification, GST Pulldown Assay, Software

39) Product Images from "Ribosomal Protein S3 Negatively Regulates Unwinding Activity of RecQ-like Helicase 4 through Their Physical Interaction *"

Article Title: Ribosomal Protein S3 Negatively Regulates Unwinding Activity of RecQ-like Helicase 4 through Their Physical Interaction *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M116.764324

RECQL4 physically interacts with RPS3. A , nuclear lysates of 293 cell lines stably expressing RECQL4-V5-his or control plasmid are used for V5 pulldown followed by anti-RPS3, anti-V5 and anti-Lamin B immunoblotting. B , Coomassie Blue-stained SDS-PAGE depicting final purified protein fraction of GST-FL-RPS3 after Biorex TM 70 and glutathione-Sepharose purification steps. C , GST pulldown assay. Anti-RECQL4 and anti-GST blots show input and pulldown fractions of GST pulldown assay carried out by incubating RECQL4-his with GST-RPS3 or GST only proteins. D , confocal images of representative U2OS cells immunostained by anti-RECQL4, anti-RPS3 antibodies, and DAPI. The plot shows nuclear RPS3 (%) and nuclear colocalization of RPS3-RECQL4 (%) quantified from three-dimensional reconstructions of Z section confocal images, using Imaris software. The data are shown as the means ± S.D. of triplicate experiments.
Figure Legend Snippet: RECQL4 physically interacts with RPS3. A , nuclear lysates of 293 cell lines stably expressing RECQL4-V5-his or control plasmid are used for V5 pulldown followed by anti-RPS3, anti-V5 and anti-Lamin B immunoblotting. B , Coomassie Blue-stained SDS-PAGE depicting final purified protein fraction of GST-FL-RPS3 after Biorex TM 70 and glutathione-Sepharose purification steps. C , GST pulldown assay. Anti-RECQL4 and anti-GST blots show input and pulldown fractions of GST pulldown assay carried out by incubating RECQL4-his with GST-RPS3 or GST only proteins. D , confocal images of representative U2OS cells immunostained by anti-RECQL4, anti-RPS3 antibodies, and DAPI. The plot shows nuclear RPS3 (%) and nuclear colocalization of RPS3-RECQL4 (%) quantified from three-dimensional reconstructions of Z section confocal images, using Imaris software. The data are shown as the means ± S.D. of triplicate experiments.

Techniques Used: Stable Transfection, Expressing, Plasmid Preparation, Staining, SDS Page, Purification, GST Pulldown Assay, Software

40) Product Images from "Cyclin-dependent kinase 5-mediated phosphorylation of chloride intracellular channel 4 promotes oxidative stress-induced neuronal death"

Article Title: Cyclin-dependent kinase 5-mediated phosphorylation of chloride intracellular channel 4 promotes oxidative stress-induced neuronal death

Journal: Cell Death & Disease

doi: 10.1038/s41419-018-0983-1

CLIC4 interacts with CDK5/p35. a Immunoblotting of proteins pulled down by glutathione Sepharose from lysates of N2a cells transiently transfected with GST alone or GST-CLIC4 as indicated. Membranes were probed with antibodies against the indicated proteins. b Immunoblotting of proteins pulled down by glutathione Sepharose from lysates of N2a cells transiently transfected with HA-CLIC4 and GST alone or GST-CLIC4. Membranes were probed with antibodies to the indicated proteins. c Immunoblotting of proteins immunoprecipitated from brain homogenates of wild-type C57BL/6 mice with antibodies against normal IgG or anti-CLIC4 antibody. d Immunoblotting of proteins immunoprecipitated from brain homogenates of wild-type C57BL/6 mice with antibodies against normal IgG, anti-CDK5 or anti-p35 antibody. e – g Immunofluorescence staining of DIV7 primary cortical neurons with anti-CDK5 and anti-CLIC4 antibodies showed the co-localization of CDK5 and CLIC4. Higher magnification around neuronal soma ( f ) and neurite ( g ) of the indicated area of e was presented and the co-localization was pointed with arrows. DAPI is a nucleus dye. Scale bar = 10 μm. h BiFC fluorescence and immunostaining showed that CDK5 and CLIC4 interacted in the cytoplasm of N2a cells. N2a cells were transfected with VN173-CDK5 and VC155 vector or VC155-CLIC4, then immunostained with anti-GFP antibody. DAPI is a nucleus dye. Scale bar = 10 μm
Figure Legend Snippet: CLIC4 interacts with CDK5/p35. a Immunoblotting of proteins pulled down by glutathione Sepharose from lysates of N2a cells transiently transfected with GST alone or GST-CLIC4 as indicated. Membranes were probed with antibodies against the indicated proteins. b Immunoblotting of proteins pulled down by glutathione Sepharose from lysates of N2a cells transiently transfected with HA-CLIC4 and GST alone or GST-CLIC4. Membranes were probed with antibodies to the indicated proteins. c Immunoblotting of proteins immunoprecipitated from brain homogenates of wild-type C57BL/6 mice with antibodies against normal IgG or anti-CLIC4 antibody. d Immunoblotting of proteins immunoprecipitated from brain homogenates of wild-type C57BL/6 mice with antibodies against normal IgG, anti-CDK5 or anti-p35 antibody. e – g Immunofluorescence staining of DIV7 primary cortical neurons with anti-CDK5 and anti-CLIC4 antibodies showed the co-localization of CDK5 and CLIC4. Higher magnification around neuronal soma ( f ) and neurite ( g ) of the indicated area of e was presented and the co-localization was pointed with arrows. DAPI is a nucleus dye. Scale bar = 10 μm. h BiFC fluorescence and immunostaining showed that CDK5 and CLIC4 interacted in the cytoplasm of N2a cells. N2a cells were transfected with VN173-CDK5 and VC155 vector or VC155-CLIC4, then immunostained with anti-GFP antibody. DAPI is a nucleus dye. Scale bar = 10 μm

Techniques Used: Transfection, Immunoprecipitation, Mouse Assay, Immunofluorescence, Staining, Bimolecular Fluorescence Complementation Assay, Fluorescence, Immunostaining, Plasmid Preparation

CDK5 promotes CLIC4 protein stability. a , b Protein levels of CLIC4 in N2a cells transiently transfected with GFP tag or GFP-p25 for 24 h. Relative CLIC4 levels were quantified in b ( n = 3 experiments). c , d CLIC4 and CDK5 protein levels in the brain homogenates of wild-type and CDK5−/− littermate embryos at E16.5. Embryos were genotyped and brain samples were analyzed by western blotting ( c ). Upper band in the agarose gel indicates CDK5−/− genotype, while lower one means wild-type. Relative CLIC4 levels were quantified in D ( n = 3 experiments, 4 embryos each group). e , f Immunoblotting of lysates of primary cortical neurons treated with 5 μM Roscovitine for 24 h. Relative CLIC4 levels were quantified in f ( n = 3 experiments). g , h Turnover of myc-CLIC4 and myc-CLIC4 (S108D) in N2a cells treated with 100 μM cycloheximide (CHX) for indicated times. Cell lysates were immunoblotted with anti-myc and anti-β-actin antibodies ( g ). Normalized myc-tagged CLIC4 proteins levels were quantified in h ( n = 3 experiments). i , j Turnover of HA-CLIC4 in N2a cells transiently transfected with HA-CLIC4/GFP-CDK5/GFP-p25 or HA-CLIC4/GFP-CDK5-KD/GFP-p25 and treated with CHX for indicated times. Lysates were subjected to western blotting for HA-tagged and GFP-tagged proteins and β-actin ( i ). Normalized HA-CLIC4 protein levels were quantified in j ( n = 3 experiments). Data are presented as the mean and SEM, and were analyzed by unpaired Student’s t -test ( b , d , f ) or two-way ANOVA test ( h , j ). * P
Figure Legend Snippet: CDK5 promotes CLIC4 protein stability. a , b Protein levels of CLIC4 in N2a cells transiently transfected with GFP tag or GFP-p25 for 24 h. Relative CLIC4 levels were quantified in b ( n = 3 experiments). c , d CLIC4 and CDK5 protein levels in the brain homogenates of wild-type and CDK5−/− littermate embryos at E16.5. Embryos were genotyped and brain samples were analyzed by western blotting ( c ). Upper band in the agarose gel indicates CDK5−/− genotype, while lower one means wild-type. Relative CLIC4 levels were quantified in D ( n = 3 experiments, 4 embryos each group). e , f Immunoblotting of lysates of primary cortical neurons treated with 5 μM Roscovitine for 24 h. Relative CLIC4 levels were quantified in f ( n = 3 experiments). g , h Turnover of myc-CLIC4 and myc-CLIC4 (S108D) in N2a cells treated with 100 μM cycloheximide (CHX) for indicated times. Cell lysates were immunoblotted with anti-myc and anti-β-actin antibodies ( g ). Normalized myc-tagged CLIC4 proteins levels were quantified in h ( n = 3 experiments). i , j Turnover of HA-CLIC4 in N2a cells transiently transfected with HA-CLIC4/GFP-CDK5/GFP-p25 or HA-CLIC4/GFP-CDK5-KD/GFP-p25 and treated with CHX for indicated times. Lysates were subjected to western blotting for HA-tagged and GFP-tagged proteins and β-actin ( i ). Normalized HA-CLIC4 protein levels were quantified in j ( n = 3 experiments). Data are presented as the mean and SEM, and were analyzed by unpaired Student’s t -test ( b , d , f ) or two-way ANOVA test ( h , j ). * P

Techniques Used: Transfection, Western Blot, Agarose Gel Electrophoresis

Related Articles

Lysis:

Article Title: Multivalent interaction of ESCO2 with the replication machinery is required for cohesion
Article Snippet: .. Co-precipitation assays GST-peptide fusions were purified in Lysis Buffer (50mM TRIS 8.2, 500mM KCl, 1 mM DTT, 1% Triton X-100) and left on Glutathione Sepharose 4B (GE Healthcare) beads following purification. ..

Incubation:

Article Title: Exportin 4 Interacts with Sox9 through the HMG Box and Inhibits the DNA Binding of Sox9
Article Snippet: .. After the incubation, the glutathione-Sepharose was washed three times with B400 buffer, washed two times with B100 buffer, and then incubated with nuclear extracts from HeLa cells at 4°C for 2 hr. .. The proteins bound to the Sepharose were eluted by lithium dodecyl sulfate (LDS) sample buffer (Invitrogen) and subjected to Western blotting analysis using anti-Exp4 antibody.

Article Title: The autophagy adaptor NDP52 and the FIP200 coiled-coil allosterically activate ULK1 complex membrane recruitment
Article Snippet: .. The supernatant was incubated with Glutathione Sepharose 4B (GE Healthcare) or Ni-NTA Resins (Qiagen) as appropriate, with gentle shaking for 2 hours at 4 °C. ..

Purification:

Article Title: Multivalent interaction of ESCO2 with the replication machinery is required for cohesion
Article Snippet: .. Co-precipitation assays GST-peptide fusions were purified in Lysis Buffer (50mM TRIS 8.2, 500mM KCl, 1 mM DTT, 1% Triton X-100) and left on Glutathione Sepharose 4B (GE Healthcare) beads following purification. ..

Article Title: C-terminal Phosphorylation of LKB1 Is Not Required for Regulation of AMP-activated Protein Kinase, BRSK1, BRSK2, or Cell Cycle Arrest *
Article Snippet: .. After purification on glutathione-Sepharose, we obtained equal yields of full-length and truncated LKB1L , and both co-purified with FLAG-STRADα and myc -MO25α as expected ( ). ..

Article Title: Interaction of two photoreceptors in the regulation of bacterial photosynthesis genes
Article Snippet: .. The purification was performed using glutathione sepharose 4B according to the manufacturer’s instruction (Amersham Biosciences, Freiburg, Germany). ..

Article Title: Novel DNA Aptamers for Parkinson’s Disease Treatment Inhibit α-Synuclein Aggregation and Facilitate its Degradation
Article Snippet: .. Then the fusion protein GST-α-syn was purified on glutathione-sepharose 4B according to the manufacturer’s instructions (GE Healthcare, Boston, MA). .. The purified GST-fusion proteins were desalted on Vivaspin 6 column from GE Healthcare, followed by dialysis into binding buffer (PBS, 1 mM MgCI2 , pH 7.4) to remove the free glutathione.

Article Title: A key role for Ctf4 in coupling the MCM2-7 helicase to DNA polymerase ? within the eukaryotic replisome
Article Snippet: .. Subsequently this was used to generate a cell extract from which we purified GINS-Ctf4 or Ctf4-Pol1NT using Glutathione Sepharose (17-0756-01, GE Healthcare), Ni-NTA Agarose (30230, Qiagen), or Strep-Tactin Superflow (2-1206-025, IBA GmbH). .. Gel filtration was performed with Akta Explorer or Pharmacia SMART systems, using Superdex 200 or Superose 6 columns.

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    GE Healthcare glutathione sepharose 4b
    α-syn Aptamers Were Selected through SELEX (A) Schematic illustration of the method used for α-syn aptamer selection. GST-tagged α-syn was immobilized on <t>glutathione-sepharose</t> beads. The ssDNA library was incubated with the target beads for binding. Unbound oligonucleotides were washed away, and the bound ones were released by heating at 95°C. The selected binders were amplified by PCR with biotinylated primers. ssDNAs were subsequently purified from the PCR product using streptavidin-coated magnetic beads, resulting in an enriched DNA pool, which was used in the next SELEX round. After the last round, the selected ssDNAs were sequenced by deep sequencing. (B) The aptamer candidates. After deep sequencing, the two sequences with most frequently appearing were selected as the aptamer candidates. (C) Aptamer binding specificity assay by dot blotting. Five microgram samples (α-syn, GST, Aβ 42 , BSA, and three domains of α-syn) were respectively immobilized onto the nitrocellulose membrane for binding of each aptamer.
    Glutathione Sepharose 4b, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 94/100, based on 2034 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Beta-1 adrenergic receptor (β1AR) binds directly to golgin-160 (1–393) . Representative gels for the purification of golgin-160 (1–393) and its binding to β1AR are shown. ( A ) The NEB IMPACT system was used to create a purified, untagged golgin-160 (1–393) following cleavage of the intein tag. DTT-induced cleavage caused enrichment of an approximately 60 kDa protein, which was specifically eluted off of the chitin column. This protein band could be detected using immunoblotting with an antibody to the N-terminus of golgin-160. Input, protein added to the chitin column; Cleaved, protein on the chitin column after addition of DTT but before elution; Eluate, protein released from the column after cleavage; *, golgin-160 (1–393) ; **, GST fusion proteins; ( B ) The purified, untagged golgin-160 head domain was incubated with purified GST or GST-β1AR L3 pre-bound to <t>glutathione-Sepharose</t> 4B beads. The beads were washed and bound golgin-160 (1–393) was detected by Coomassie blue staining after SDS-PAGE. Note that the samples in panel A were run on a 4%–12% gradient gel, whereas those in B were run on a 10% gel.
    Glutathione Sepharose 4b Beads, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 94/100, based on 113 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare glutathione sepharose resin
    The OAR of Rtf1 interacts directly with the CTR of Spt5. (A) Recombinant GST (pGEX-3X), GST-Rtf1-His 6 (pAP21), GST-Rtf1ΔOAR-His 6 (pMM26), and GST-OAR (pMM25) proteins, bound to <t>glutathione-Sepharose</t> beads, were incubated with the same amount of
    Glutathione Sepharose Resin, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 94/100, based on 198 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    α-syn Aptamers Were Selected through SELEX (A) Schematic illustration of the method used for α-syn aptamer selection. GST-tagged α-syn was immobilized on glutathione-sepharose beads. The ssDNA library was incubated with the target beads for binding. Unbound oligonucleotides were washed away, and the bound ones were released by heating at 95°C. The selected binders were amplified by PCR with biotinylated primers. ssDNAs were subsequently purified from the PCR product using streptavidin-coated magnetic beads, resulting in an enriched DNA pool, which was used in the next SELEX round. After the last round, the selected ssDNAs were sequenced by deep sequencing. (B) The aptamer candidates. After deep sequencing, the two sequences with most frequently appearing were selected as the aptamer candidates. (C) Aptamer binding specificity assay by dot blotting. Five microgram samples (α-syn, GST, Aβ 42 , BSA, and three domains of α-syn) were respectively immobilized onto the nitrocellulose membrane for binding of each aptamer.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Novel DNA Aptamers for Parkinson’s Disease Treatment Inhibit α-Synuclein Aggregation and Facilitate its Degradation

    doi: 10.1016/j.omtn.2018.02.011

    Figure Lengend Snippet: α-syn Aptamers Were Selected through SELEX (A) Schematic illustration of the method used for α-syn aptamer selection. GST-tagged α-syn was immobilized on glutathione-sepharose beads. The ssDNA library was incubated with the target beads for binding. Unbound oligonucleotides were washed away, and the bound ones were released by heating at 95°C. The selected binders were amplified by PCR with biotinylated primers. ssDNAs were subsequently purified from the PCR product using streptavidin-coated magnetic beads, resulting in an enriched DNA pool, which was used in the next SELEX round. After the last round, the selected ssDNAs were sequenced by deep sequencing. (B) The aptamer candidates. After deep sequencing, the two sequences with most frequently appearing were selected as the aptamer candidates. (C) Aptamer binding specificity assay by dot blotting. Five microgram samples (α-syn, GST, Aβ 42 , BSA, and three domains of α-syn) were respectively immobilized onto the nitrocellulose membrane for binding of each aptamer.

    Article Snippet: Then the fusion protein GST-α-syn was purified on glutathione-sepharose 4B according to the manufacturer’s instructions (GE Healthcare, Boston, MA).

    Techniques: Selection, Incubation, Binding Assay, Amplification, Polymerase Chain Reaction, Purification, Magnetic Beads, Sequencing

    Effect of C-terminal truncation of LKB1 on AMPK activation in cell-free assays and ACC phosphorylation and cell cycle progress in G361 melanoma cells. A , plasmids encoding GST fusions of wild type LKB1 L and a C-terminal truncation (1–343) were co-expressed with FLAG-STRADα and myc -MO25α in HEK-293 cells and purified on glutathione-Sepharose. The purified products were analyzed by Western blotting using anti-GST, anti-FLAG, and anti- myc antibodies. B , a bacterially expressed GST fusion of the AMPK-α1 kinase domain was incubated with MgATP and various concentrations of GST-LKB1·FLAG-STRADα· myc -MO25α complex purified as in A , and AMPK activity was determined after 15 min. C , phosphorylation of the AMPK target, ACC, total ACC, and expression of GFP-LKB1 assessed using an anti-GFP antibody, in G361 cells co-expressing STRADα and MO25α with free GFP (control) or GFP fusions of wild type LKB1L and a C-terminally truncated mutant (1–343). D , cell cycle analysis of GFP-expressing cells treated as in Fig. 5 C , 18 h after nocodazole treatment.

    Journal: The Journal of Biological Chemistry

    Article Title: C-terminal Phosphorylation of LKB1 Is Not Required for Regulation of AMP-activated Protein Kinase, BRSK1, BRSK2, or Cell Cycle Arrest *

    doi: 10.1074/jbc.M806152200

    Figure Lengend Snippet: Effect of C-terminal truncation of LKB1 on AMPK activation in cell-free assays and ACC phosphorylation and cell cycle progress in G361 melanoma cells. A , plasmids encoding GST fusions of wild type LKB1 L and a C-terminal truncation (1–343) were co-expressed with FLAG-STRADα and myc -MO25α in HEK-293 cells and purified on glutathione-Sepharose. The purified products were analyzed by Western blotting using anti-GST, anti-FLAG, and anti- myc antibodies. B , a bacterially expressed GST fusion of the AMPK-α1 kinase domain was incubated with MgATP and various concentrations of GST-LKB1·FLAG-STRADα· myc -MO25α complex purified as in A , and AMPK activity was determined after 15 min. C , phosphorylation of the AMPK target, ACC, total ACC, and expression of GFP-LKB1 assessed using an anti-GFP antibody, in G361 cells co-expressing STRADα and MO25α with free GFP (control) or GFP fusions of wild type LKB1L and a C-terminally truncated mutant (1–343). D , cell cycle analysis of GFP-expressing cells treated as in Fig. 5 C , 18 h after nocodazole treatment.

    Article Snippet: After purification on glutathione-Sepharose, we obtained equal yields of full-length and truncated LKB1L , and both co-purified with FLAG-STRADα and myc -MO25α as expected ( ).

    Techniques: Activation Assay, Purification, Western Blot, Incubation, Activity Assay, Expressing, Mutagenesis, Cell Cycle Assay

    Phosphorylation and activation of AMPK, BRSK1, and BRSK2 by LKB1 variants in cell-free assays. A , purification of LKB1·STRADα·MO25α complexes from HEK-293 cells. Plasmids encoding FLAG-tagged STRADα and myc -tagged MO25α were co-expressed in HEK-293 cells with the indicated variants of GST-tagged LKB1. GST fusions were purified on glutathione-Sepharose, and the products were analyzed by Western blotting using anti-GST, anti-FLAG, or anti- myc antibodies. B –E, bacterially expressed GST fusions with the kinase domains of AMPK-α1 ( B and C ), BRSK1 ( D ), or BRSK2 ( E ) were incubated with MgATP and LKB1·STRADα·MO25α complexes (50 μg·ml –1 ) purified as in A . After 15 min the incubations were analyzed for activity of AMPK ( B ), BRSK1 ( D ), or BRSK2 ( E ) and for phosphorylation of the threonine residue equivalent to Thr-172 using anti-pT172 antibody ( C –E). WT , wild type.

    Journal: The Journal of Biological Chemistry

    Article Title: C-terminal Phosphorylation of LKB1 Is Not Required for Regulation of AMP-activated Protein Kinase, BRSK1, BRSK2, or Cell Cycle Arrest *

    doi: 10.1074/jbc.M806152200

    Figure Lengend Snippet: Phosphorylation and activation of AMPK, BRSK1, and BRSK2 by LKB1 variants in cell-free assays. A , purification of LKB1·STRADα·MO25α complexes from HEK-293 cells. Plasmids encoding FLAG-tagged STRADα and myc -tagged MO25α were co-expressed in HEK-293 cells with the indicated variants of GST-tagged LKB1. GST fusions were purified on glutathione-Sepharose, and the products were analyzed by Western blotting using anti-GST, anti-FLAG, or anti- myc antibodies. B –E, bacterially expressed GST fusions with the kinase domains of AMPK-α1 ( B and C ), BRSK1 ( D ), or BRSK2 ( E ) were incubated with MgATP and LKB1·STRADα·MO25α complexes (50 μg·ml –1 ) purified as in A . After 15 min the incubations were analyzed for activity of AMPK ( B ), BRSK1 ( D ), or BRSK2 ( E ) and for phosphorylation of the threonine residue equivalent to Thr-172 using anti-pT172 antibody ( C –E). WT , wild type.

    Article Snippet: After purification on glutathione-Sepharose, we obtained equal yields of full-length and truncated LKB1L , and both co-purified with FLAG-STRADα and myc -MO25α as expected ( ).

    Techniques: Activation Assay, Purification, Western Blot, Incubation, Activity Assay

    Beta-1 adrenergic receptor (β1AR) binds directly to golgin-160 (1–393) . Representative gels for the purification of golgin-160 (1–393) and its binding to β1AR are shown. ( A ) The NEB IMPACT system was used to create a purified, untagged golgin-160 (1–393) following cleavage of the intein tag. DTT-induced cleavage caused enrichment of an approximately 60 kDa protein, which was specifically eluted off of the chitin column. This protein band could be detected using immunoblotting with an antibody to the N-terminus of golgin-160. Input, protein added to the chitin column; Cleaved, protein on the chitin column after addition of DTT but before elution; Eluate, protein released from the column after cleavage; *, golgin-160 (1–393) ; **, GST fusion proteins; ( B ) The purified, untagged golgin-160 head domain was incubated with purified GST or GST-β1AR L3 pre-bound to glutathione-Sepharose 4B beads. The beads were washed and bound golgin-160 (1–393) was detected by Coomassie blue staining after SDS-PAGE. Note that the samples in panel A were run on a 4%–12% gradient gel, whereas those in B were run on a 10% gel.

    Journal: International Journal of Molecular Sciences

    Article Title: Three Basic Residues of Intracellular Loop 3 of the Beta-1 Adrenergic Receptor Are Required for Golgin-160-Dependent Trafficking

    doi: 10.3390/ijms15022929

    Figure Lengend Snippet: Beta-1 adrenergic receptor (β1AR) binds directly to golgin-160 (1–393) . Representative gels for the purification of golgin-160 (1–393) and its binding to β1AR are shown. ( A ) The NEB IMPACT system was used to create a purified, untagged golgin-160 (1–393) following cleavage of the intein tag. DTT-induced cleavage caused enrichment of an approximately 60 kDa protein, which was specifically eluted off of the chitin column. This protein band could be detected using immunoblotting with an antibody to the N-terminus of golgin-160. Input, protein added to the chitin column; Cleaved, protein on the chitin column after addition of DTT but before elution; Eluate, protein released from the column after cleavage; *, golgin-160 (1–393) ; **, GST fusion proteins; ( B ) The purified, untagged golgin-160 head domain was incubated with purified GST or GST-β1AR L3 pre-bound to glutathione-Sepharose 4B beads. The beads were washed and bound golgin-160 (1–393) was detected by Coomassie blue staining after SDS-PAGE. Note that the samples in panel A were run on a 4%–12% gradient gel, whereas those in B were run on a 10% gel.

    Article Snippet: The soluble fraction of the lysed cells was incubated 2 h at 4 °C with 10 μg GST alone or GST-tagged golgin-160(1–393) that had been pre-conjugated to glutathione-Sepharose 4B beads.

    Techniques: Purification, Binding Assay, Incubation, Staining, SDS Page

    The OAR of Rtf1 interacts directly with the CTR of Spt5. (A) Recombinant GST (pGEX-3X), GST-Rtf1-His 6 (pAP21), GST-Rtf1ΔOAR-His 6 (pMM26), and GST-OAR (pMM25) proteins, bound to glutathione-Sepharose beads, were incubated with the same amount of

    Journal: Molecular and Cellular Biology

    Article Title: The Recruitment of the Saccharomyces cerevisiae Paf1 Complex to Active Genes Requires a Domain of Rtf1 That Directly Interacts with the Spt4-Spt5 Complex

    doi: 10.1128/MCB.00270-13

    Figure Lengend Snippet: The OAR of Rtf1 interacts directly with the CTR of Spt5. (A) Recombinant GST (pGEX-3X), GST-Rtf1-His 6 (pAP21), GST-Rtf1ΔOAR-His 6 (pMM26), and GST-OAR (pMM25) proteins, bound to glutathione-Sepharose beads, were incubated with the same amount of

    Article Snippet: Clarified lysates were incubated with 1 ml of bovine serum albumin (BSA)-blocked 50% glutathione-Sepharose resin (GE Healthcare) for 1 h at 4°C to purify GST and GST-OAR.

    Techniques: Recombinant, Incubation