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    Structured Review

    Millipore immunoprecipitates
    Fig. 9. Co-immunoprecipitation of RNA Pol II and p38. HeLa cells were transfected with either pCMV5 or pCMV5-Flag-p38. Transfected cells were serum starved and, when indicated, stimulated with 0.3 M NaCl for 20 min. Lysates from the transfected cells were immunoprecipitated with anti-RNA Pol II antibody (8WG16), and the <t>immunoprecipitates</t> were subjected to immunoblot analysis for flag-p38 using the anti-Flag M2 monoclonal antibody.
    Immunoprecipitates, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 615 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Osmostress-induced transcription by Hot1 depends on a Hog1-mediated recruitment of the RNA Pol II"

    Article Title: Osmostress-induced transcription by Hot1 depends on a Hog1-mediated recruitment of the RNA Pol II

    Journal: The EMBO Journal

    doi: 10.1093/emboj/cdg243

    Fig. 9. Co-immunoprecipitation of RNA Pol II and p38. HeLa cells were transfected with either pCMV5 or pCMV5-Flag-p38. Transfected cells were serum starved and, when indicated, stimulated with 0.3 M NaCl for 20 min. Lysates from the transfected cells were immunoprecipitated with anti-RNA Pol II antibody (8WG16), and the immunoprecipitates were subjected to immunoblot analysis for flag-p38 using the anti-Flag M2 monoclonal antibody.
    Figure Legend Snippet: Fig. 9. Co-immunoprecipitation of RNA Pol II and p38. HeLa cells were transfected with either pCMV5 or pCMV5-Flag-p38. Transfected cells were serum starved and, when indicated, stimulated with 0.3 M NaCl for 20 min. Lysates from the transfected cells were immunoprecipitated with anti-RNA Pol II antibody (8WG16), and the immunoprecipitates were subjected to immunoblot analysis for flag-p38 using the anti-Flag M2 monoclonal antibody.

    Techniques Used: Immunoprecipitation, Transfection

    2) Product Images from "An Oncogenic Protein Golgi Phosphoprotein 3 Up-regulates Cell Migration via Sialylation *"

    Article Title: An Oncogenic Protein Golgi Phosphoprotein 3 Up-regulates Cell Migration via Sialylation *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.542688

    Alteration of N -glycosylation in GOLPH3-knockdown cells. A, total expression levels of β1 integrin were analyzed by Western blotting ( WB ). The same amounts of cell lysates (200 μg) were obtained from control and KD cells, which were picked up from HeLa cells expressed as shRNA for control ( Ctrl ) and GOLPH3 (KD1,2) using the Phoenix system. Cell lysates were immunoprecipitated ( IP ) with anti-β1 antibody. The immunoprecipitates of β1 integrin were treated with (+) or without peptide: N -glycosidase F ( PNGase ) (−), and then immunoblotted with anti-β1 antibody ( upper panel ). The knockdown efficiency of GOLPH3 was confirmed by immunoblotting with anti-GOLPH3 antibody ( middle panel ). The α-tubulin was used as a loading control to warrant the same amounts of proteins to be used ( lower panel ). B, analysis of PA- N -glycans was by reversed-phase HPLC. Then N -glycans released from control or KD cells with peptide: N -glycosidase F were pyridylaminated as described under “Experimental Procedures.” The PA- N -glycans ( upper panel ), sequentially digested with sialidase ( middle panel ) and β-galactosidase ( lower panel ), were subjected to reversed phase HPLC. The asterisk indicates the peaks for sialylated N -glycans. C, RT-PCR for mRNA expression of several sialyltransferases and sialidase as indicated. The β-actin was used as a loading control. Ctrl, control shRNA; KD , shRNA for GOLPH3 -knockdown; β 1, integrin β1.
    Figure Legend Snippet: Alteration of N -glycosylation in GOLPH3-knockdown cells. A, total expression levels of β1 integrin were analyzed by Western blotting ( WB ). The same amounts of cell lysates (200 μg) were obtained from control and KD cells, which were picked up from HeLa cells expressed as shRNA for control ( Ctrl ) and GOLPH3 (KD1,2) using the Phoenix system. Cell lysates were immunoprecipitated ( IP ) with anti-β1 antibody. The immunoprecipitates of β1 integrin were treated with (+) or without peptide: N -glycosidase F ( PNGase ) (−), and then immunoblotted with anti-β1 antibody ( upper panel ). The knockdown efficiency of GOLPH3 was confirmed by immunoblotting with anti-GOLPH3 antibody ( middle panel ). The α-tubulin was used as a loading control to warrant the same amounts of proteins to be used ( lower panel ). B, analysis of PA- N -glycans was by reversed-phase HPLC. Then N -glycans released from control or KD cells with peptide: N -glycosidase F were pyridylaminated as described under “Experimental Procedures.” The PA- N -glycans ( upper panel ), sequentially digested with sialidase ( middle panel ) and β-galactosidase ( lower panel ), were subjected to reversed phase HPLC. The asterisk indicates the peaks for sialylated N -glycans. C, RT-PCR for mRNA expression of several sialyltransferases and sialidase as indicated. The β-actin was used as a loading control. Ctrl, control shRNA; KD , shRNA for GOLPH3 -knockdown; β 1, integrin β1.

    Techniques Used: Expressing, Western Blot, shRNA, Immunoprecipitation, High Performance Liquid Chromatography, Reverse Transcription Polymerase Chain Reaction

    3) Product Images from "SUV39H1 interacts with HTLV-1 Tax and abrogates Tax transactivation of HTLV-1 LTR"

    Article Title: SUV39H1 interacts with HTLV-1 Tax and abrogates Tax transactivation of HTLV-1 LTR

    Journal: Retrovirology

    doi: 10.1186/1742-4690-3-5

    Results of in vitro methyltransferase assays. (a) Time course analysis. Top panel shows a representative fluorogram of the reaction mixtures at the indicated time points analyzed by 15% SDS-PAGE. The middle panel shows the relative levels of methylation measured by densitometric analyses of the bands. Bottom panel, a result of immunoblot analysis of transduced SUV39H1 by anti-SUV39H1 monoclonal antibody, showing comparable levels of SUV39H1 expression in each sample. (b) A representative result of three independent experiments of in vitro methyltransferase assays of SUV39H1 transduced with or without Tax. The reaction time was 30 min. The second panel shows the relative intensities of the methylated H3 bands. Lower panels show the results of immunoblot analyses of the immunoprecipitates and whole cell lysates to show the presence of SUV39H1 with or without Tax. IP, immunoprecipitation; IB, immunoblot. Antibodies used are indicated on the side of the panels.
    Figure Legend Snippet: Results of in vitro methyltransferase assays. (a) Time course analysis. Top panel shows a representative fluorogram of the reaction mixtures at the indicated time points analyzed by 15% SDS-PAGE. The middle panel shows the relative levels of methylation measured by densitometric analyses of the bands. Bottom panel, a result of immunoblot analysis of transduced SUV39H1 by anti-SUV39H1 monoclonal antibody, showing comparable levels of SUV39H1 expression in each sample. (b) A representative result of three independent experiments of in vitro methyltransferase assays of SUV39H1 transduced with or without Tax. The reaction time was 30 min. The second panel shows the relative intensities of the methylated H3 bands. Lower panels show the results of immunoblot analyses of the immunoprecipitates and whole cell lysates to show the presence of SUV39H1 with or without Tax. IP, immunoprecipitation; IB, immunoblot. Antibodies used are indicated on the side of the panels.

    Techniques Used: In Vitro, SDS Page, Methylation, Expressing, Transduction, Immunoprecipitation

    Tax interacts with SUV39H1 in vitro. (a) HEK293T cells were transiently cotransfected with GST-SUV39H1 or GST and Tax. After 48 h, the cells were lysed and the proteins were affinity purified with Glutathione Sepharose 4B. Purified proteins were separated by SDS-PAGE, transferred to a PVDF membrane, and probed with anti-Tax antibody Lt-4 (top panel). Expression of transduced proteins was confirmed by immunoblot analyses of whole cell lysates using respective antibodies (lower panels). (b) HEK293T cells were transiently co-transfected with expression plasmids, GST-SUV39H1 or GST and Tax. After 48 h, the cells were lysed and the proteins were immunoprecipitated with Lt-4. The immunoprecipitates were separated by SDS-PAGE, transferred to a PVDF membrane, and probed with anti-SUV39H1 or anti-GST antibody (upper panels). Expression of proteins was confirmed by immunoblot analyses of whole cell lysates using respective antibodies (lower panels). (c) Direct interaction between SUV39H1 and Tax. Bacterially expressed GST-SUV39H1 and GST were purified with Glutathione Sepharose 4B, and histidine-tagged wild type Tax (His-Tax) was purified with ProBond Resin (Promega). GST-SUV39H1 and GST were bound to Glutathione Sepharose 4B, and mixed with purified His-Tax in PBS. After centrifugation, proteins bound to Glutathione Sepharose 4B were separated by electrophoresis, transferred to a PVDF membrane, and probed with anti-Tax antibody. As a control, an aliquot of purified His-Tax was run in lane 4. IP, immunoprecipitation; IB, immunoblot; H.C., heavy chain
    Figure Legend Snippet: Tax interacts with SUV39H1 in vitro. (a) HEK293T cells were transiently cotransfected with GST-SUV39H1 or GST and Tax. After 48 h, the cells were lysed and the proteins were affinity purified with Glutathione Sepharose 4B. Purified proteins were separated by SDS-PAGE, transferred to a PVDF membrane, and probed with anti-Tax antibody Lt-4 (top panel). Expression of transduced proteins was confirmed by immunoblot analyses of whole cell lysates using respective antibodies (lower panels). (b) HEK293T cells were transiently co-transfected with expression plasmids, GST-SUV39H1 or GST and Tax. After 48 h, the cells were lysed and the proteins were immunoprecipitated with Lt-4. The immunoprecipitates were separated by SDS-PAGE, transferred to a PVDF membrane, and probed with anti-SUV39H1 or anti-GST antibody (upper panels). Expression of proteins was confirmed by immunoblot analyses of whole cell lysates using respective antibodies (lower panels). (c) Direct interaction between SUV39H1 and Tax. Bacterially expressed GST-SUV39H1 and GST were purified with Glutathione Sepharose 4B, and histidine-tagged wild type Tax (His-Tax) was purified with ProBond Resin (Promega). GST-SUV39H1 and GST were bound to Glutathione Sepharose 4B, and mixed with purified His-Tax in PBS. After centrifugation, proteins bound to Glutathione Sepharose 4B were separated by electrophoresis, transferred to a PVDF membrane, and probed with anti-Tax antibody. As a control, an aliquot of purified His-Tax was run in lane 4. IP, immunoprecipitation; IB, immunoblot; H.C., heavy chain

    Techniques Used: In Vitro, Affinity Purification, Purification, SDS Page, Expressing, Transfection, Immunoprecipitation, Centrifugation, Electrophoresis

    4) Product Images from "The p38 SAPK Is Recruited to Chromatin via Its Interaction with Transcription Factors *"

    Article Title: The p38 SAPK Is Recruited to Chromatin via Its Interaction with Transcription Factors *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.155846

    p38 SAPK recruitment at the c-Fos promoter requires the transcription factor Elk1. A , HeLa cells were transfected with either control or Elk1 siRNAs and the cell lysates were inmunoprecipitated with anti-Elk1 antibody. Both inputs and immunoprecipitates were analyzed by Western blotting with anti-Elk1 antibody. * marks a nonspecific band. B , HeLa cells were transfected with the indicated siRNAs and treated with 100 m m NaCl for 2 h. The mRNA levels of c-Fos, Elk1, and GAPDH were analyzed by PCR. C , HeLa cells were treated with 100 m m NaCl for 60 min either in the presence or absence SB203580 and subjected to ChIP assay using an anti-Elk1 antibody. D , HeLa cells were transfected with pCDNA3–3HA-p38α along with control or Elk1 siRNAs. Cells were treated with 100 m m NaCl for 60 min and subjected to ChIP assay with anti-HA antibody. Immunoprecipitated DNA fragments were subjected to PCR analysis.
    Figure Legend Snippet: p38 SAPK recruitment at the c-Fos promoter requires the transcription factor Elk1. A , HeLa cells were transfected with either control or Elk1 siRNAs and the cell lysates were inmunoprecipitated with anti-Elk1 antibody. Both inputs and immunoprecipitates were analyzed by Western blotting with anti-Elk1 antibody. * marks a nonspecific band. B , HeLa cells were transfected with the indicated siRNAs and treated with 100 m m NaCl for 2 h. The mRNA levels of c-Fos, Elk1, and GAPDH were analyzed by PCR. C , HeLa cells were treated with 100 m m NaCl for 60 min either in the presence or absence SB203580 and subjected to ChIP assay using an anti-Elk1 antibody. D , HeLa cells were transfected with pCDNA3–3HA-p38α along with control or Elk1 siRNAs. Cells were treated with 100 m m NaCl for 60 min and subjected to ChIP assay with anti-HA antibody. Immunoprecipitated DNA fragments were subjected to PCR analysis.

    Techniques Used: Transfection, Western Blot, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Immunoprecipitation

    5) Product Images from "Mto2 multisite phosphorylation inactivates non-spindle microtubule nucleation complexes during mitosis"

    Article Title: Mto2 multisite phosphorylation inactivates non-spindle microtubule nucleation complexes during mitosis

    Journal: Nature Communications

    doi: 10.1038/ncomms8929

    Mto2 phosphomutants can maintain Mto1–Mto2 and Mto1/2-γ-TuC interactions in mitosis. ( a ) Abundance ratios of Mto2[17A] non-phosphopeptides in interphase versus mitosis, as measured by SILAC mass spectrometry, indicating that several Mto2[17A] regions undergo high-stoichiometry modifications during mitosis. Grey bars indicate quantified peptides. Red lines indicate position of residues already mutated in Mto2[17A] (see Fig. 3a ). Brackets indicate regions containing additional phosphosite mutations in the second-generation series of mto2-phosphomutant cells shown in ( b ). ( b ) Anti-Mto2 western blot of extracts from mto2[17A] , wild-type ( mto2 + ) and second-generation mto2-phosphomutant cells, containing additional phosphosite mutations beyond those in mto2[17A] . Second-generation phosphomutants are named according to the region containing additional mutations, with the exception of the mutant corresponding to NT2, which is named mto2[24A] . Mto2 expression levels are relative to interphase mto2 + cells (third lane, set to 100; see Methods). Cold-sensitive nda3-KM311 β-tubulin allele was used to arrest cells in metaphase. I, interphase; M, mitosis. ( c ) Anti-Mto1, anti-Mto2 and Anti-Gtb1 western blots of cell extracts and corresponding IgG pulldowns, from interphase and metaphase-arrested wild-type and mto2-phosphomutant cells expressing Mto1-SZZ. Graphs show quantification of Mto2 (or phosphomutant Mto2) and Gtb1 copurifying with Mto1-SZZ in the pulldowns above, normalized to wild-type (WT) interphase cells (first column; see Methods). cs , cold-sensitive nda3-KM311 β-tubulin allele, used for metaphase arrest. ( d ) Anti-Mto2 Western blots of cell extracts and anti-GFP immunoprecipitates from untagged ( mto2 + ), mto2-GFP , mto2[17A]-GFP and mto2[24A]-GFP cells; immunoprecipitates were either treated with lambda phosphatase (PPase, +) or untreated (−). Upper panels show proteins resolved on 10% Laemmli SDS–PAGE. Mto2-GFP expression levels are relative to cells expressing untagged Mto2 (Lane 1, set to 100; see Methods). Lower panel shows proteins resolved on 6% Phos-tag SDS–PAGE; because protein migration on Phos-tag gels is strongly affected by phosphorylation state, molecular weight markers here are only fiducial. Aspect ratio in lower panel was altered relative to original image, to match width of upper panel. All mto2-GFP strains are also nda3-KM311 .
    Figure Legend Snippet: Mto2 phosphomutants can maintain Mto1–Mto2 and Mto1/2-γ-TuC interactions in mitosis. ( a ) Abundance ratios of Mto2[17A] non-phosphopeptides in interphase versus mitosis, as measured by SILAC mass spectrometry, indicating that several Mto2[17A] regions undergo high-stoichiometry modifications during mitosis. Grey bars indicate quantified peptides. Red lines indicate position of residues already mutated in Mto2[17A] (see Fig. 3a ). Brackets indicate regions containing additional phosphosite mutations in the second-generation series of mto2-phosphomutant cells shown in ( b ). ( b ) Anti-Mto2 western blot of extracts from mto2[17A] , wild-type ( mto2 + ) and second-generation mto2-phosphomutant cells, containing additional phosphosite mutations beyond those in mto2[17A] . Second-generation phosphomutants are named according to the region containing additional mutations, with the exception of the mutant corresponding to NT2, which is named mto2[24A] . Mto2 expression levels are relative to interphase mto2 + cells (third lane, set to 100; see Methods). Cold-sensitive nda3-KM311 β-tubulin allele was used to arrest cells in metaphase. I, interphase; M, mitosis. ( c ) Anti-Mto1, anti-Mto2 and Anti-Gtb1 western blots of cell extracts and corresponding IgG pulldowns, from interphase and metaphase-arrested wild-type and mto2-phosphomutant cells expressing Mto1-SZZ. Graphs show quantification of Mto2 (or phosphomutant Mto2) and Gtb1 copurifying with Mto1-SZZ in the pulldowns above, normalized to wild-type (WT) interphase cells (first column; see Methods). cs , cold-sensitive nda3-KM311 β-tubulin allele, used for metaphase arrest. ( d ) Anti-Mto2 Western blots of cell extracts and anti-GFP immunoprecipitates from untagged ( mto2 + ), mto2-GFP , mto2[17A]-GFP and mto2[24A]-GFP cells; immunoprecipitates were either treated with lambda phosphatase (PPase, +) or untreated (−). Upper panels show proteins resolved on 10% Laemmli SDS–PAGE. Mto2-GFP expression levels are relative to cells expressing untagged Mto2 (Lane 1, set to 100; see Methods). Lower panel shows proteins resolved on 6% Phos-tag SDS–PAGE; because protein migration on Phos-tag gels is strongly affected by phosphorylation state, molecular weight markers here are only fiducial. Aspect ratio in lower panel was altered relative to original image, to match width of upper panel. All mto2-GFP strains are also nda3-KM311 .

    Techniques Used: Mass Spectrometry, Western Blot, Mutagenesis, Expressing, SDS Page, Migration, Molecular Weight

    6) Product Images from "Coactivator-Dependent Acetylation Stabilizes Members of the SREBP Family of Transcription Factors"

    Article Title: Coactivator-Dependent Acetylation Stabilizes Members of the SREBP Family of Transcription Factors

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.23.7.2587-2599.2003

    SREBPs are substrates for p300 and CBP. (A) Lysates from HeLa cells grown in lipoprotein-deficient media were immunoprecipitated with anti-Gal4 (lane 1) or anti-SREBP1a (lane 2) antibodies. The immunoprecipitates were washed, and the proteins were separated by SDS-PAGE. Coimmunoprecipitated p300 was detected by Western blotting using anti-p300 antibodies. The amount of mature SREBP1a in the immunoprecipitates was determined with anti-SREBP1a antibodies. (B) HepG2 cells were transfected with SYNSRE-luc in the absence (lane 1) or presence (lanes 2 to 10) of Flag-tagged SREBP1a and in the absence or presence of p300 (25 to 250 ng), either the wild type (lanes 3 to 6) or the ΔHAT1 mutant (lanes 7 to 10). Thirty-six hours after transfection, the luciferase activity was measured. The data represent the averages ± standard deviations of three independent experiments performed in duplicate. (C) His-tagged SREBP1a and SREBP2 were subjected to in vitro acetylation in the absence (lanes 2 and 4) or presence (lanes 3 and 5) of p300 as described in Materials and Methods. After the proteins were separated by SDS-PAGE, the gel was dried and analyzed by phosphorimage analysis. (D) Flag-tagged SREBP1a (lanes 1 to 4) and SREBP2 (lanes 5 to 8) were expressed in 293T cells in the absence or presence of the indicated acetyltransferases. Following immunoprecipitation with anti-Flag antibodies, the samples were resolved by SDS-PAGE and the acetylation of the SREBPs was determined with anti-acetyl lysine antibodies (α-AcK). The levels of SREBPs in the immunoprecipitates were determined with anti-Flag antibodies (α-Flag). The levels of Gal4-p300, Gal4-CBP, and Flag-P/CAF in the cell lysates were determined by Western blotting.
    Figure Legend Snippet: SREBPs are substrates for p300 and CBP. (A) Lysates from HeLa cells grown in lipoprotein-deficient media were immunoprecipitated with anti-Gal4 (lane 1) or anti-SREBP1a (lane 2) antibodies. The immunoprecipitates were washed, and the proteins were separated by SDS-PAGE. Coimmunoprecipitated p300 was detected by Western blotting using anti-p300 antibodies. The amount of mature SREBP1a in the immunoprecipitates was determined with anti-SREBP1a antibodies. (B) HepG2 cells were transfected with SYNSRE-luc in the absence (lane 1) or presence (lanes 2 to 10) of Flag-tagged SREBP1a and in the absence or presence of p300 (25 to 250 ng), either the wild type (lanes 3 to 6) or the ΔHAT1 mutant (lanes 7 to 10). Thirty-six hours after transfection, the luciferase activity was measured. The data represent the averages ± standard deviations of three independent experiments performed in duplicate. (C) His-tagged SREBP1a and SREBP2 were subjected to in vitro acetylation in the absence (lanes 2 and 4) or presence (lanes 3 and 5) of p300 as described in Materials and Methods. After the proteins were separated by SDS-PAGE, the gel was dried and analyzed by phosphorimage analysis. (D) Flag-tagged SREBP1a (lanes 1 to 4) and SREBP2 (lanes 5 to 8) were expressed in 293T cells in the absence or presence of the indicated acetyltransferases. Following immunoprecipitation with anti-Flag antibodies, the samples were resolved by SDS-PAGE and the acetylation of the SREBPs was determined with anti-acetyl lysine antibodies (α-AcK). The levels of SREBPs in the immunoprecipitates were determined with anti-Flag antibodies (α-Flag). The levels of Gal4-p300, Gal4-CBP, and Flag-P/CAF in the cell lysates were determined by Western blotting.

    Techniques Used: Immunoprecipitation, SDS Page, Western Blot, Transfection, Mutagenesis, Luciferase, Activity Assay, In Vitro

    Acetylation of SREBP1a prevents its ubiquitination. (A) Flag-tagged wild-type SREBP1a and the K333R mutant were expressed in 293T cells in the absence or presence of HA-ubiquitin. Thirty-six hours after transfection, SREBP1a was immunoprecipitated from cell lysates and resolved by SDS-PAGE. The ubiquitination of SREBP1a was determined by Western blotting using anti-HA antibodies (α-HA). The migration of molecular mass standards (in kilodaltons) is indicated to the right. The levels of SREBP1a in the immunoprecipitates were determined by Western blotting using anti-Flag antibodies (α-Flag). (B) GST-SREBP1a was used as the substrate in in vitro ubiquitination reactions in the absence or presence of rabbit reticulocyte lysate (RRL). Where indicated, GST-SREBP1a was acetylated prior to the ubiquitination reaction (lane 3). The samples were resolved by SDS-PAGE, and the ubiquitination of SREBP1a was determined with antiubiquitin antibodies (α-UB). The migration of molecular mass standards (in kilodaltons) is indicated to the right. The amount of SREBP1a and its acetylation were determined with anti-SREBP1a (α-SRE1) and anti-acetyl lysine (α-AcK) antibodies, respectively.
    Figure Legend Snippet: Acetylation of SREBP1a prevents its ubiquitination. (A) Flag-tagged wild-type SREBP1a and the K333R mutant were expressed in 293T cells in the absence or presence of HA-ubiquitin. Thirty-six hours after transfection, SREBP1a was immunoprecipitated from cell lysates and resolved by SDS-PAGE. The ubiquitination of SREBP1a was determined by Western blotting using anti-HA antibodies (α-HA). The migration of molecular mass standards (in kilodaltons) is indicated to the right. The levels of SREBP1a in the immunoprecipitates were determined by Western blotting using anti-Flag antibodies (α-Flag). (B) GST-SREBP1a was used as the substrate in in vitro ubiquitination reactions in the absence or presence of rabbit reticulocyte lysate (RRL). Where indicated, GST-SREBP1a was acetylated prior to the ubiquitination reaction (lane 3). The samples were resolved by SDS-PAGE, and the ubiquitination of SREBP1a was determined with antiubiquitin antibodies (α-UB). The migration of molecular mass standards (in kilodaltons) is indicated to the right. The amount of SREBP1a and its acetylation were determined with anti-SREBP1a (α-SRE1) and anti-acetyl lysine (α-AcK) antibodies, respectively.

    Techniques Used: Mutagenesis, Transfection, Immunoprecipitation, SDS Page, Western Blot, Migration, In Vitro

    SREBP1a is acetylated in its core DNA-binding domain by p300. (A) Flag-tagged wild-type (WT) SREBP1a and the indicated lysine mutants were expressed in 293T cells in the absence or presence of p300-HA. Following immunoprecipitation of SREBP1a, samples were resolved by SDS-PAGE and the acetylation of SREBP1a was detected with anti-acetyl lysine antibodies (α-AcK). The amount of SREBP1a in the immunoprecipitates was determined with anti-Flag antibodies (α-Flag). The levels of p300-HA in the cell lysates were determined by Western blotting (α-HA). (B) Increasing amounts of in vitro-translated Flag-SREBP1a, the wild type and the indicated mutants, were used in EMSAs with a 32 P-labeled probe containing the SRE-1 sequence from the LDLR promoter. (C) Cos7 cells were transfected with SYNSRE-luc in the absence (lane 1) or presence (lanes 2 to 16) of increasing amounts (0.5 to 2.5 ng) of Flag-tagged SREBP1a, the wild type and the indicated mutants. Thirty-six hours after transfection, the luciferase activity was measured. The data represent the averages ± standard deviations (SD) of three independent experiments performed in duplicate. (D) HepG2 cells were transfected with SYNSRE-luc in the absence or presence of Flag-tagged SREBP1a, the wild type and the K333R mutant, in the absence or presence of p300 (25 or 100 ng). Thirty-six hours after transfection, the luciferase activity was measured. The data represent the averages ± SD of three independent experiments performed in duplicate.
    Figure Legend Snippet: SREBP1a is acetylated in its core DNA-binding domain by p300. (A) Flag-tagged wild-type (WT) SREBP1a and the indicated lysine mutants were expressed in 293T cells in the absence or presence of p300-HA. Following immunoprecipitation of SREBP1a, samples were resolved by SDS-PAGE and the acetylation of SREBP1a was detected with anti-acetyl lysine antibodies (α-AcK). The amount of SREBP1a in the immunoprecipitates was determined with anti-Flag antibodies (α-Flag). The levels of p300-HA in the cell lysates were determined by Western blotting (α-HA). (B) Increasing amounts of in vitro-translated Flag-SREBP1a, the wild type and the indicated mutants, were used in EMSAs with a 32 P-labeled probe containing the SRE-1 sequence from the LDLR promoter. (C) Cos7 cells were transfected with SYNSRE-luc in the absence (lane 1) or presence (lanes 2 to 16) of increasing amounts (0.5 to 2.5 ng) of Flag-tagged SREBP1a, the wild type and the indicated mutants. Thirty-six hours after transfection, the luciferase activity was measured. The data represent the averages ± standard deviations (SD) of three independent experiments performed in duplicate. (D) HepG2 cells were transfected with SYNSRE-luc in the absence or presence of Flag-tagged SREBP1a, the wild type and the K333R mutant, in the absence or presence of p300 (25 or 100 ng). Thirty-six hours after transfection, the luciferase activity was measured. The data represent the averages ± SD of three independent experiments performed in duplicate.

    Techniques Used: Binding Assay, Immunoprecipitation, SDS Page, Western Blot, In Vitro, Labeling, Sequencing, Transfection, Luciferase, Activity Assay, Mutagenesis

    7) Product Images from "Targeted Disruption of Fibulin-4 Abolishes Elastogenesis and Causes Perinatal Lethality in Mice"

    Article Title: Targeted Disruption of Fibulin-4 Abolishes Elastogenesis and Causes Perinatal Lethality in Mice

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.26.5.1700-1709.2006

    Fibulin-4 interacts with tropoelastin. (A) Purified recombinant mouse fibulin-4. A total of 1 μg of purified fibulin-4 was used for the Coomassie blue-stained gel, and 10 ng of the protein was used for the Western blot. An anti-FLAG antibody was used for immunodetection. A single band with the correct mass for fibulin-4 was detected by both Coomassie blue staining and immunoblotting. (B) Solid-phase binding assay using recombinant fibulin-4 as a soluble ligand. Note that fibulin-4 binds to tropoelastin in the presence of Ca 2+ but that the binding is inhibited in the presence of EDTA. Data were obtained as the results of triplicate experiments, and values shown are means ± standard deviations. (C) Coimmunoprecipitation of fibulin-4 with tropoelastin. Lanes 1 to 4, antitropoelastin blotting of immunoprecipitates from lysates of 293T cells transfected with both tropoelastin and fibulin-4 (lanes 1 and 3) or tropoelastin alone (lanes 2 and 4). Tropoelastin was detected in the immunoprecipitate by anti-fibulin-4 (7B9) (lane 3). Lanes 5 to 8, anti-fibulin-4 (11E2) blotting of immunoprecipitates from lysates of 293T cells transfected with both tropoelastin and fibulin-4 (lanes 5 and 7) or fibulin-4 alone (lanes 6 and 8). Fibulin-4 was detected in the immunoprecipitate by antitropoelastin (lane 7). IP, immunoprecipitation; TE, tropoelastin; fb4, fibulin-4.
    Figure Legend Snippet: Fibulin-4 interacts with tropoelastin. (A) Purified recombinant mouse fibulin-4. A total of 1 μg of purified fibulin-4 was used for the Coomassie blue-stained gel, and 10 ng of the protein was used for the Western blot. An anti-FLAG antibody was used for immunodetection. A single band with the correct mass for fibulin-4 was detected by both Coomassie blue staining and immunoblotting. (B) Solid-phase binding assay using recombinant fibulin-4 as a soluble ligand. Note that fibulin-4 binds to tropoelastin in the presence of Ca 2+ but that the binding is inhibited in the presence of EDTA. Data were obtained as the results of triplicate experiments, and values shown are means ± standard deviations. (C) Coimmunoprecipitation of fibulin-4 with tropoelastin. Lanes 1 to 4, antitropoelastin blotting of immunoprecipitates from lysates of 293T cells transfected with both tropoelastin and fibulin-4 (lanes 1 and 3) or tropoelastin alone (lanes 2 and 4). Tropoelastin was detected in the immunoprecipitate by anti-fibulin-4 (7B9) (lane 3). Lanes 5 to 8, anti-fibulin-4 (11E2) blotting of immunoprecipitates from lysates of 293T cells transfected with both tropoelastin and fibulin-4 (lanes 5 and 7) or fibulin-4 alone (lanes 6 and 8). Fibulin-4 was detected in the immunoprecipitate by antitropoelastin (lane 7). IP, immunoprecipitation; TE, tropoelastin; fb4, fibulin-4.

    Techniques Used: Purification, Recombinant, Staining, Western Blot, Immunodetection, Binding Assay, Transfection, Immunoprecipitation

    8) Product Images from "A PGC-1α-O-GlcNAc Transferase Complex Regulates FoxO Transcription Factor Activity in Response to Glucose"

    Article Title: A PGC-1α-O-GlcNAc Transferase Complex Regulates FoxO Transcription Factor Activity in Response to Glucose

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M808890200

    PGC-1α interacts with OGT. A , Fao cells were infected with Ad-FLAG-PGC-1α and treated with insulin for 1 h prior to harvesting and OGT co-immunoprecipitation ( IP ). Immunoprecipitates were subjected to SDS-PAGE and immunoblotted ( IB ) for the presence of PGC-1α. Membranes were then stripped and blotted for OGT. Immunoblots are representative of three experiments. B , Fao cells were infected with Ad-FLAG-PGC-1α and treated with insulin for 1 h prior to harvesting and FLAG co-immunoprecipitation. Immunoprecipitates were subjected to SDS-PAGE and immunoblotted for the presence of OGT. Membranes were then stripped and blotted for PGC-1α. C , gel filtration chromatography (SMART system, Superdex 200 column, phosphate-buffered saline, 1% Nonidet P-40 buffer) of lysates from rat liver incubated on ice for 30 min with either normal IgG or anti-OGT antibodies (AL28). Fractions were subjected to SDS-PAGE and blotted using anti-PGC-1α, anti-OGT (DM-17), and anti CPB. Data are representative of two experiments.
    Figure Legend Snippet: PGC-1α interacts with OGT. A , Fao cells were infected with Ad-FLAG-PGC-1α and treated with insulin for 1 h prior to harvesting and OGT co-immunoprecipitation ( IP ). Immunoprecipitates were subjected to SDS-PAGE and immunoblotted ( IB ) for the presence of PGC-1α. Membranes were then stripped and blotted for OGT. Immunoblots are representative of three experiments. B , Fao cells were infected with Ad-FLAG-PGC-1α and treated with insulin for 1 h prior to harvesting and FLAG co-immunoprecipitation. Immunoprecipitates were subjected to SDS-PAGE and immunoblotted for the presence of OGT. Membranes were then stripped and blotted for PGC-1α. C , gel filtration chromatography (SMART system, Superdex 200 column, phosphate-buffered saline, 1% Nonidet P-40 buffer) of lysates from rat liver incubated on ice for 30 min with either normal IgG or anti-OGT antibodies (AL28). Fractions were subjected to SDS-PAGE and blotted using anti-PGC-1α, anti-OGT (DM-17), and anti CPB. Data are representative of two experiments.

    Techniques Used: Pyrolysis Gas Chromatography, Infection, Immunoprecipitation, SDS Page, Western Blot, Filtration, Chromatography, Incubation

    9) Product Images from "The Prp19 WD40 Domain Contains a Conserved Protein Interaction Region Essential for its Function"

    Article Title: The Prp19 WD40 Domain Contains a Conserved Protein Interaction Region Essential for its Function

    Journal: Structure (London, England : 1993)

    doi: 10.1016/j.str.2010.02.015

    Cwc2/Cwf2 is present in the NTC in a 1:2 ratio with Prp19/Cwf8. A) An anti-myc immunoblot of an IgG immunoprecipitate from either a wild-type S. pombe strain (KGY246) or from h− cdc5-TAP cwf8-Myc 13 cwf2-Myc 13 ade6-M210 leu1-32 ura4-D18 (KGY7136). B) Anti-Myc (upper panel) and anti-GFP immunoblots of immunoprecipitates (IP) from cwf2-Myc 13 , cwf2-GFP , and cwf2-Myc 13 cwf2-GFP strains. Immunoprecipitations were performed with anti-Myc or anti-GFP antibodies.
    Figure Legend Snippet: Cwc2/Cwf2 is present in the NTC in a 1:2 ratio with Prp19/Cwf8. A) An anti-myc immunoblot of an IgG immunoprecipitate from either a wild-type S. pombe strain (KGY246) or from h− cdc5-TAP cwf8-Myc 13 cwf2-Myc 13 ade6-M210 leu1-32 ura4-D18 (KGY7136). B) Anti-Myc (upper panel) and anti-GFP immunoblots of immunoprecipitates (IP) from cwf2-Myc 13 , cwf2-GFP , and cwf2-Myc 13 cwf2-GFP strains. Immunoprecipitations were performed with anti-Myc or anti-GFP antibodies.

    Techniques Used: Western Blot

    10) Product Images from "BCL-3 promotes a cancer stem cell phenotype by enhancing β-catenin signalling in colorectal tumour cells"

    Article Title: BCL-3 promotes a cancer stem cell phenotype by enhancing β-catenin signalling in colorectal tumour cells

    Journal: Disease Models & Mechanisms

    doi: 10.1242/dmm.037697

    BCL-3 interacts with β-catenin in CRC cell lines. (A,B) BCL-3 Co-IP performed in SW1463 nuclear-enriched lysates. Unbound (immuno-depleted) lysates show depletion of proteins after IP. Immunoprecipitates were analysed by western blot for BCL-3 and β-catenin. IgG serves as a negative control. (A) CYLD serves as a positive control for BCL-3 binding. (B) Cells were treated with 100 ng/ml TNF-α for 6 h prior to lysis. Immunoprecipitates were additionally analysed for p52. Note the presence of a non-specific (ns) band in BCL-3 western analysis with use of Abcam anti-BCL-3 antibody. (C,D) Nuclear β-catenin Co-IPs in SW620 cells. Panels C and D represent experimental replicates. Western analysis of non-treated and 6-h TNF-α-treated cells following β-catenin Co-IP. TCF4 serves as a positive control for β-catenin binding. Mouse pan-IgG serves as a negative control. Unbound, immuno-depleted lysates following Co-IP; WB, western blot antibody.
    Figure Legend Snippet: BCL-3 interacts with β-catenin in CRC cell lines. (A,B) BCL-3 Co-IP performed in SW1463 nuclear-enriched lysates. Unbound (immuno-depleted) lysates show depletion of proteins after IP. Immunoprecipitates were analysed by western blot for BCL-3 and β-catenin. IgG serves as a negative control. (A) CYLD serves as a positive control for BCL-3 binding. (B) Cells were treated with 100 ng/ml TNF-α for 6 h prior to lysis. Immunoprecipitates were additionally analysed for p52. Note the presence of a non-specific (ns) band in BCL-3 western analysis with use of Abcam anti-BCL-3 antibody. (C,D) Nuclear β-catenin Co-IPs in SW620 cells. Panels C and D represent experimental replicates. Western analysis of non-treated and 6-h TNF-α-treated cells following β-catenin Co-IP. TCF4 serves as a positive control for β-catenin binding. Mouse pan-IgG serves as a negative control. Unbound, immuno-depleted lysates following Co-IP; WB, western blot antibody.

    Techniques Used: Co-Immunoprecipitation Assay, Western Blot, Negative Control, Positive Control, Binding Assay, Lysis

    11) Product Images from "Dissociation of FAK/p130CAS/c-Src Complex during Mitosis: Role of Mitosis-specific Serine Phosphorylation of FAK "

    Article Title: Dissociation of FAK/p130CAS/c-Src Complex during Mitosis: Role of Mitosis-specific Serine Phosphorylation of FAK

    Journal: The Journal of Cell Biology

    doi:

    Tyrosine rephosphorylation of FAK (a), paxillin (b), and CAS (c) during post-mitotic cell spreading. FAK, CAS, and paxillin were immunoprecipitated from interphase cells (I), mitotic cells (M), and cells released from mitotic arrest (numbered lanes, n = min removed from nocodazole). The immunoprecipitates were transferred to PVDF membranes, first immunoblotted with PY20, then reprobed with the antibodies against FAK, paxillin, and CAS. The levels of phosphotyrosine are shown by ratios (100% for interphase level) of the levels of PY20 reactivities divided by the levels of FAK, paxillin, or CAS. Note that FAK exhibits the fastest recovery of tyrosine rephosphorylation as well as the greatest increase in tyrosine phosphorylation during 80– 180 min after the release of mitotic arrest.
    Figure Legend Snippet: Tyrosine rephosphorylation of FAK (a), paxillin (b), and CAS (c) during post-mitotic cell spreading. FAK, CAS, and paxillin were immunoprecipitated from interphase cells (I), mitotic cells (M), and cells released from mitotic arrest (numbered lanes, n = min removed from nocodazole). The immunoprecipitates were transferred to PVDF membranes, first immunoblotted with PY20, then reprobed with the antibodies against FAK, paxillin, and CAS. The levels of phosphotyrosine are shown by ratios (100% for interphase level) of the levels of PY20 reactivities divided by the levels of FAK, paxillin, or CAS. Note that FAK exhibits the fastest recovery of tyrosine rephosphorylation as well as the greatest increase in tyrosine phosphorylation during 80– 180 min after the release of mitotic arrest.

    Techniques Used: Immunoprecipitation

    Serine phosphorylation is responsible for the mobility shifts. (a) Loss of mobility shifts by the treatment with PP1. FAK, CAS, and paxillin (PAX) were immunoprecipitated under condition I from mitotic (odd numbered lanes) or from interphase (even numbered lanes) cells, and half of the immunoprecipitates (lanes 3, 4, 7, 8, 11, and 12) were treated with PP1 to dephosphorylate phosphoserine and phosphothreonine. Note that PP1 treatment eliminates or greatly decreases the mobility shifts shown by mitotic FAK (compare lanes 1 and 3), CAS (compare lanes 5 and 7), and paxillin (compare lanes 9 and 11). (b) Tyrosine dephosphorylation of FAK, CAS, and paxillin during mitosis. FAK, CAS, and paxillin (PAX) were again immunoprecipitated under condition I from mitotic (lanes M) and interphase (lanes I) cells. The immunoprecipitates were first immunoblotted with PY20 (top; lanes 1, 2, 5, 6, 9, and 10), and then reprobed with FAK, CAS, and paxillin antibodies (bottom; lanes 3, 4, 7, 8, 11, and 12). Note that PY20 reacts strongly with the immunoprecipitates prepared from interphase but not from mitotic cells, indicating that mitotic FAK, CAS, and paxillin are dephosphorylated at tyrosine residues. (c) Phosphate incorporation of FAK, CAS, and paxillin. FAK, CAS, and paxillin were immunoprecipitated under condition I from mitotic (lanes M) and interphase (lanes I) cells that had been labeled in vivo with 32 P inorganic phosphate. The 32 P-labeled immunoprecipitates were analyzed by SDS-PAGE followed by autoradiography. The levels of 32 P incorporation in mitotic FAK, CAS, and paxillin are not greatly increased because serine phosphorylation is negated by tyrosine dephosphorylation.
    Figure Legend Snippet: Serine phosphorylation is responsible for the mobility shifts. (a) Loss of mobility shifts by the treatment with PP1. FAK, CAS, and paxillin (PAX) were immunoprecipitated under condition I from mitotic (odd numbered lanes) or from interphase (even numbered lanes) cells, and half of the immunoprecipitates (lanes 3, 4, 7, 8, 11, and 12) were treated with PP1 to dephosphorylate phosphoserine and phosphothreonine. Note that PP1 treatment eliminates or greatly decreases the mobility shifts shown by mitotic FAK (compare lanes 1 and 3), CAS (compare lanes 5 and 7), and paxillin (compare lanes 9 and 11). (b) Tyrosine dephosphorylation of FAK, CAS, and paxillin during mitosis. FAK, CAS, and paxillin (PAX) were again immunoprecipitated under condition I from mitotic (lanes M) and interphase (lanes I) cells. The immunoprecipitates were first immunoblotted with PY20 (top; lanes 1, 2, 5, 6, 9, and 10), and then reprobed with FAK, CAS, and paxillin antibodies (bottom; lanes 3, 4, 7, 8, 11, and 12). Note that PY20 reacts strongly with the immunoprecipitates prepared from interphase but not from mitotic cells, indicating that mitotic FAK, CAS, and paxillin are dephosphorylated at tyrosine residues. (c) Phosphate incorporation of FAK, CAS, and paxillin. FAK, CAS, and paxillin were immunoprecipitated under condition I from mitotic (lanes M) and interphase (lanes I) cells that had been labeled in vivo with 32 P inorganic phosphate. The 32 P-labeled immunoprecipitates were analyzed by SDS-PAGE followed by autoradiography. The levels of 32 P incorporation in mitotic FAK, CAS, and paxillin are not greatly increased because serine phosphorylation is negated by tyrosine dephosphorylation.

    Techniques Used: Immunoprecipitation, De-Phosphorylation Assay, Labeling, In Vivo, SDS Page, Autoradiography

    Reconstitution of a FAK/CAS complex following dephosphorylation of mitotic FAK. (a) FAK was immunoprecipitated under condition I from mitotic cells and divided into three equal parts for further treatment: lane 1, neither incubated with interphase extracts nor treated with PP1; lane 2, incubated with interphase extracts following treatment with PP1; lane 3, incubated with interphase extracts. After extensive washing, the association of FAK with CAS was examined by blotting with the anti-FAK antibody or anti-CAS antibody. Phosphotyrosine levels of FAK were examined by PY20. For comparison, FAK immunoprecipitates from interphase cells were blotted with the same antibodies (lane 4). (b) Quantitative analyses. The CAS reassociation and phosphotyrosine levels of FAK are expressed as ratios to those found in FAK incubated with interphase extracts but without prior PP1 treatment (−PP1). Data were obtained from five independent experiments.
    Figure Legend Snippet: Reconstitution of a FAK/CAS complex following dephosphorylation of mitotic FAK. (a) FAK was immunoprecipitated under condition I from mitotic cells and divided into three equal parts for further treatment: lane 1, neither incubated with interphase extracts nor treated with PP1; lane 2, incubated with interphase extracts following treatment with PP1; lane 3, incubated with interphase extracts. After extensive washing, the association of FAK with CAS was examined by blotting with the anti-FAK antibody or anti-CAS antibody. Phosphotyrosine levels of FAK were examined by PY20. For comparison, FAK immunoprecipitates from interphase cells were blotted with the same antibodies (lane 4). (b) Quantitative analyses. The CAS reassociation and phosphotyrosine levels of FAK are expressed as ratios to those found in FAK incubated with interphase extracts but without prior PP1 treatment (−PP1). Data were obtained from five independent experiments.

    Techniques Used: De-Phosphorylation Assay, Immunoprecipitation, Incubation

    12) Product Images from "Hemidesmosome Formation Is Initiated by the ?4 Integrin Subunit, Requires Complex Formation of ?4 and HD1/Plectin, and Involves a Direct Interaction between ?4 and the Bullous Pemphigoid Antigen 180 "

    Article Title: Hemidesmosome Formation Is Initiated by the ?4 Integrin Subunit, Requires Complex Formation of ?4 and HD1/Plectin, and Involves a Direct Interaction between ?4 and the Bullous Pemphigoid Antigen 180

    Journal: The Journal of Cell Biology

    doi:

    Coimmunoprecipi-tation of β 4 and BP180. Lysates of COS-7 cells cotransfected with cDNAs for α 6A and wild-type β 4A, β 4 1,355 , or β 4 1,328 as well as an empty pCI-Neo vector or a pCI-Neo construct encoding the BP180 cytoplasmic domain were subjected to immunoprecipitation with a mixture (1:1:1) of three anti- β 4 mAbs, 4.3E1, 113C and 450-9D, respectively, or with the mAb FLAG™ M2. Samples were resolved on a SDS-polyacrylamide (8%) gel under nonreducing conditions. Shown is an immunoblot analysis developed with the rabbit polyclonal anti-serum against β 4 ( top ) and the mAb FLAG™ M2 to detect BP180 ( bottom ) among the immunoprecipitated proteins. When samples immunoprecipitated with the anti- β 4 mAbs were subjected to immunoblotting with the mAb FLAG™ M2 ( bottom ) or a polyclonal anti-BP180 antiserum (data not shown), the mutant form of BP180 was not detectable in the anti- β 4 immunoprecipitates. The positions of molecular weight standards (in kD) are indicated on the right.
    Figure Legend Snippet: Coimmunoprecipi-tation of β 4 and BP180. Lysates of COS-7 cells cotransfected with cDNAs for α 6A and wild-type β 4A, β 4 1,355 , or β 4 1,328 as well as an empty pCI-Neo vector or a pCI-Neo construct encoding the BP180 cytoplasmic domain were subjected to immunoprecipitation with a mixture (1:1:1) of three anti- β 4 mAbs, 4.3E1, 113C and 450-9D, respectively, or with the mAb FLAG™ M2. Samples were resolved on a SDS-polyacrylamide (8%) gel under nonreducing conditions. Shown is an immunoblot analysis developed with the rabbit polyclonal anti-serum against β 4 ( top ) and the mAb FLAG™ M2 to detect BP180 ( bottom ) among the immunoprecipitated proteins. When samples immunoprecipitated with the anti- β 4 mAbs were subjected to immunoblotting with the mAb FLAG™ M2 ( bottom ) or a polyclonal anti-BP180 antiserum (data not shown), the mutant form of BP180 was not detectable in the anti- β 4 immunoprecipitates. The positions of molecular weight standards (in kD) are indicated on the right.

    Techniques Used: Plasmid Preparation, Construct, Immunoprecipitation, Mutagenesis, Molecular Weight

    Immunoprecipitation of integrin complexes and BP180 from NHK and PA-JEB keratinocytes. Lysates of 125 I-labeled NHK ( left ) and PA-JEB keratinocytes ( right ) were immunoprecipitated with the mAbs P1E6 (against α2 , lane 1 ), J143 ( α3 , lane 2 ), Sam-1 ( α5 , lane 3 ), NKI-M9 ( αv , lane 4 ), J8H ( α6 , lane 5 ), TS2/16 ( β1 , lane 6 ), 450-9D ( β4 , lane 7 ), 439-9B ( β4 , lane 8 , right ) and 1D1 ( BP180 , lane 8 , left and lane 9 , right , respectively). The antibody against α6 precipitated this subunit associated with β4 from NHK, whereas from the PA-JEB cells α6 and β1 were precipitated, but not β4. The faint band which migrates just above β1 and seen in the lanes containing the anti-β4 immunoprecipitates from PA-JEB cells, represents a nonspecific product. β1 is found in association with α2, α3, α5, and α6 in PA-JEB cells, but only with α2, α3, and α5 in NHK. Precipitation of β1 with α5 is evident after prolonged exposure (data not shown). Samples were analyzed on a SDS-polyacrylamide (5%) gel under nonreducing conditions. The positions of molecular weight standards (in kD) are indicated on the left.
    Figure Legend Snippet: Immunoprecipitation of integrin complexes and BP180 from NHK and PA-JEB keratinocytes. Lysates of 125 I-labeled NHK ( left ) and PA-JEB keratinocytes ( right ) were immunoprecipitated with the mAbs P1E6 (against α2 , lane 1 ), J143 ( α3 , lane 2 ), Sam-1 ( α5 , lane 3 ), NKI-M9 ( αv , lane 4 ), J8H ( α6 , lane 5 ), TS2/16 ( β1 , lane 6 ), 450-9D ( β4 , lane 7 ), 439-9B ( β4 , lane 8 , right ) and 1D1 ( BP180 , lane 8 , left and lane 9 , right , respectively). The antibody against α6 precipitated this subunit associated with β4 from NHK, whereas from the PA-JEB cells α6 and β1 were precipitated, but not β4. The faint band which migrates just above β1 and seen in the lanes containing the anti-β4 immunoprecipitates from PA-JEB cells, represents a nonspecific product. β1 is found in association with α2, α3, α5, and α6 in PA-JEB cells, but only with α2, α3, and α5 in NHK. Precipitation of β1 with α5 is evident after prolonged exposure (data not shown). Samples were analyzed on a SDS-polyacrylamide (5%) gel under nonreducing conditions. The positions of molecular weight standards (in kD) are indicated on the left.

    Techniques Used: Immunoprecipitation, Labeling, Molecular Weight

    13) Product Images from "Skap2 is required for β2 integrin–mediated neutrophil recruitment and functions"

    Article Title: Skap2 is required for β2 integrin–mediated neutrophil recruitment and functions

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20160647

    Cross talk of Skap2 and WASp is indispensable for β 2 integrin activation and neutrophil recruitment. (A–C) IVM of TNF-inflamed postcapillary venules of WT, Was −/− , and Skap2 −/− Was −/− mice. Rolling velocities (A), number of adherent cells (B), and number of extravasated cells (C) 2 h after TNF application are shown. n = 4 mice/group. (D) Adhesion of neutrophils in postcapillary venules of WT, Was −/− , and Skap2 −/− Was −/− mice after i.v. injection of CXCL1. n = 4 mice/group. (E and F) Soluble ICAM-1 binding (E) and soluble fibrinogen binding (F) of CXCL1-stimulated WT, Was −/− , and Skap2 −/− Was −/− neutrophils. n = 3. (G) Immunoprecipitation of Skap2 in unstimulated WT neutrophils or neutrophils plated on E-selectin with shear or stimulated with CXCL1 in solution. Immunoprecipitates were immunoblotted with anti-WASp and anti-Skap2 antibody. Input was with anti–α-tubulin antibody. n = 3. (H) WT neutrophils were left unstimulated, plated on E-selectin with shear, or stimulated with CXCL1 in solution. Lysates were incubated with GST alone (control), GST fusion proteins of the Skap2 CC, PH, or SH3 domains, or full-length Skap2. Precipitates were immunoblotted with anti-WASp, and input was with anti–α-tubulin antibody. n = 3. (I and J) In vitro co-purification of His-WASp by different Skap2 GST fusion proteins. Precipitates were immunoblotted with anti-His or anti-GST, and input controls were with anti-GST antibody. n = 3. (K–N) WT and Skap2 −/− or Was −/− neutrophils were left unstimulated, plated on E-selectin with shear, or stimulated with CXCL1 in solution. Lysates were immunoprecipitated with anti-WASp (K and L) or anti-Skap2 (M and N) antibody followed by immunoblotting with anti-phosphotyrosine (4G10), anti-WASp, or anti-Skap2 antibody. Input was immunoblotted with anti–α-tubulin antibody. Quantification is shown on the right. n = 3. *, P
    Figure Legend Snippet: Cross talk of Skap2 and WASp is indispensable for β 2 integrin activation and neutrophil recruitment. (A–C) IVM of TNF-inflamed postcapillary venules of WT, Was −/− , and Skap2 −/− Was −/− mice. Rolling velocities (A), number of adherent cells (B), and number of extravasated cells (C) 2 h after TNF application are shown. n = 4 mice/group. (D) Adhesion of neutrophils in postcapillary venules of WT, Was −/− , and Skap2 −/− Was −/− mice after i.v. injection of CXCL1. n = 4 mice/group. (E and F) Soluble ICAM-1 binding (E) and soluble fibrinogen binding (F) of CXCL1-stimulated WT, Was −/− , and Skap2 −/− Was −/− neutrophils. n = 3. (G) Immunoprecipitation of Skap2 in unstimulated WT neutrophils or neutrophils plated on E-selectin with shear or stimulated with CXCL1 in solution. Immunoprecipitates were immunoblotted with anti-WASp and anti-Skap2 antibody. Input was with anti–α-tubulin antibody. n = 3. (H) WT neutrophils were left unstimulated, plated on E-selectin with shear, or stimulated with CXCL1 in solution. Lysates were incubated with GST alone (control), GST fusion proteins of the Skap2 CC, PH, or SH3 domains, or full-length Skap2. Precipitates were immunoblotted with anti-WASp, and input was with anti–α-tubulin antibody. n = 3. (I and J) In vitro co-purification of His-WASp by different Skap2 GST fusion proteins. Precipitates were immunoblotted with anti-His or anti-GST, and input controls were with anti-GST antibody. n = 3. (K–N) WT and Skap2 −/− or Was −/− neutrophils were left unstimulated, plated on E-selectin with shear, or stimulated with CXCL1 in solution. Lysates were immunoprecipitated with anti-WASp (K and L) or anti-Skap2 (M and N) antibody followed by immunoblotting with anti-phosphotyrosine (4G10), anti-WASp, or anti-Skap2 antibody. Input was immunoblotted with anti–α-tubulin antibody. Quantification is shown on the right. n = 3. *, P

    Techniques Used: Activation Assay, Mouse Assay, Injection, Binding Assay, Immunoprecipitation, Incubation, In Vitro, Copurification

    14) Product Images from "Multiple Transmembrane Amino Acid Requirements Suggest a Highly Specific Interaction between the Bovine Papillomavirus E5 Oncoprotein and the Platelet-Derived Growth Factor Beta Receptor"

    Article Title: Multiple Transmembrane Amino Acid Requirements Suggest a Highly Specific Interaction between the Bovine Papillomavirus E5 Oncoprotein and the Platelet-Derived Growth Factor Beta Receptor

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.16.7976-7986.2002

    Biochemical and functional analysis of PDGFβR mutants containing extracellular proximal transmembrane amino acid substitutions. Each of the PDGFβR mutants containing an extracellular proximal transmembrane amino acid substitution, the wild-type PDGFβR (PR), or no exogenous receptor (LXSN) was expressed in Ba/F3 cells with (+) or without (−) E5 or v- sis as described in Materials and Methods. (A) PDGFβR was immunoprecipitated (PRIP) from cell extracts and subjected to antiphosphotyrosine (PY) or anti-PDGFβR (PR) immunoblotting to detect receptor activation and total receptor levels, respectively. (B) The E5 protein with any associated proteins was immunoprecipitated (E5IP) from cell extracts and subjected to anti-PDGFβR (PR) or antiphosphotyrosine (PY) immunoblotting to assess physical complex formation between the E5 protein and the PDGFβR. Anti-E5 (E5) immunoblotting was performed to detect E5 protein expression levels. In these experiments, the presence of the 165-kDa precursor form of the PDGFβR in E5 immunoprecipitates is indicative of complex formation between the E5 protein and the PDGFβR. Arrows to the right mark the mature (m) and precursor (p) isoforms of each receptor as well as the E5 protein. Each lane represents approximately 585 μg (A) or 675 μg (B) of extracted protein.
    Figure Legend Snippet: Biochemical and functional analysis of PDGFβR mutants containing extracellular proximal transmembrane amino acid substitutions. Each of the PDGFβR mutants containing an extracellular proximal transmembrane amino acid substitution, the wild-type PDGFβR (PR), or no exogenous receptor (LXSN) was expressed in Ba/F3 cells with (+) or without (−) E5 or v- sis as described in Materials and Methods. (A) PDGFβR was immunoprecipitated (PRIP) from cell extracts and subjected to antiphosphotyrosine (PY) or anti-PDGFβR (PR) immunoblotting to detect receptor activation and total receptor levels, respectively. (B) The E5 protein with any associated proteins was immunoprecipitated (E5IP) from cell extracts and subjected to anti-PDGFβR (PR) or antiphosphotyrosine (PY) immunoblotting to assess physical complex formation between the E5 protein and the PDGFβR. Anti-E5 (E5) immunoblotting was performed to detect E5 protein expression levels. In these experiments, the presence of the 165-kDa precursor form of the PDGFβR in E5 immunoprecipitates is indicative of complex formation between the E5 protein and the PDGFβR. Arrows to the right mark the mature (m) and precursor (p) isoforms of each receptor as well as the E5 protein. Each lane represents approximately 585 μg (A) or 675 μg (B) of extracted protein.

    Techniques Used: Functional Assay, Immunoprecipitation, Activation Assay, Expressing

    15) Product Images from "Role of MMP-2 in the Regulation of IL-6/Stat3 Survival Signaling via Interaction With ?5?1 Integrin in glioma"

    Article Title: Role of MMP-2 in the Regulation of IL-6/Stat3 Survival Signaling via Interaction With ?5?1 Integrin in glioma

    Journal: Oncogene

    doi: 10.1038/onc.2012.52

    Abrogation of IL-6/Stat3 activation by α5β1 integrin function blocking glioma xenograft cells. A, Effect of Fibronectin adhesion induced α5β1 signaling activation on pM-inhibited Stat3 activation. The 4910 and 5310 cells were transfected with mock, pSV and pM for 24 hours as described in Materials and Methods. Cells were detached from culture plates by trypsinization and re-plated on FN-coated plates and cultured for another 24 hours. Whole cell lysates were subjected western blotting and representative blots showing expression levels of phospho-Stat3, CyclinD1 and c-Myc were obtained from three individual repetitions. B, ChIP assay was performed using ChIP-IT™ Express Magnetic Chromatin Immunoprecipitation kit following manufacturer’s protocol (Active motif) and immunoprecipitated DNA reverse cross-linked, purified and subjected to PCR to detect the Stat3 recruitment at CyclinD1 and c-Myc promoter sequences where pre-immunoprecipitated input samples were used for GAPDH PCR amplification and normal sheep IgG (Nsp-IgG) immunoprecipitates was loaded as negative control. C, Cells were treated with mock, pSV, pM, rhMMP-2 and α5β1 blocking antibody for 48 hours as described in Materials and Methods and whole cell lysates were subjected to western blotting to determine the expression levels of IL-6, phospho-Stat3, CyclinD1 and c-Myc. Blots were stripped and re-probed with GAPDH to confirm equal loading.
    Figure Legend Snippet: Abrogation of IL-6/Stat3 activation by α5β1 integrin function blocking glioma xenograft cells. A, Effect of Fibronectin adhesion induced α5β1 signaling activation on pM-inhibited Stat3 activation. The 4910 and 5310 cells were transfected with mock, pSV and pM for 24 hours as described in Materials and Methods. Cells were detached from culture plates by trypsinization and re-plated on FN-coated plates and cultured for another 24 hours. Whole cell lysates were subjected western blotting and representative blots showing expression levels of phospho-Stat3, CyclinD1 and c-Myc were obtained from three individual repetitions. B, ChIP assay was performed using ChIP-IT™ Express Magnetic Chromatin Immunoprecipitation kit following manufacturer’s protocol (Active motif) and immunoprecipitated DNA reverse cross-linked, purified and subjected to PCR to detect the Stat3 recruitment at CyclinD1 and c-Myc promoter sequences where pre-immunoprecipitated input samples were used for GAPDH PCR amplification and normal sheep IgG (Nsp-IgG) immunoprecipitates was loaded as negative control. C, Cells were treated with mock, pSV, pM, rhMMP-2 and α5β1 blocking antibody for 48 hours as described in Materials and Methods and whole cell lysates were subjected to western blotting to determine the expression levels of IL-6, phospho-Stat3, CyclinD1 and c-Myc. Blots were stripped and re-probed with GAPDH to confirm equal loading.

    Techniques Used: Activation Assay, Blocking Assay, Transfection, Cell Culture, Western Blot, Expressing, Chromatin Immunoprecipitation, Immunoprecipitation, Purification, Polymerase Chain Reaction, Amplification, Negative Control

    Effect of siRNA-insensitive MMP-2 overexpression and rhMMP-2 supplementation on pM-inhibited IL-6/Stat3 signaling activation. A, After 48 hours of treatment with mock, pSV, pM, pM-FL-A141G, pSV+pM-FL-A141G, pM+pM-FL-A141G, rhMMP-2, pSV+rhMMP-2, and pM+rhMMP-2 as described in Materials and Methods, whole cell lysates were subjected to Western blotting. Blots were representative of at least three independent repetitions and GAPDH probing confirmed equal loading. B, Effect of MMP-2 overexpression by siRNA-insensitive pM-FL-A141G treatment or rhMMP-2 supplementation on pM-inhibited MMP-2/α5β1 integrin binding. Whole cell lysates were (200 µg each) were immunoprecipitated with anti-α5β1 integrin antibody (10 µL each sample) using µMACS™ protein G microbeads and MACS separation columns and immunoprecipitates were subjected to Western blotting. The mock sample immunoprecipitated with non-specific IgG was loaded as the negative control. Input shows whole cell lysate (50 µg) of mock sample served as the positive control. Blots were probed with anti-MMP-2 antibody and representative blots from three independent repetitions were shown. Adjacent bar diagram shows the densitometric analyses of the relative band intensity represented as mean±SE values obtained from three repetitions. The significant differences among various treatment groups were indicated by *at p
    Figure Legend Snippet: Effect of siRNA-insensitive MMP-2 overexpression and rhMMP-2 supplementation on pM-inhibited IL-6/Stat3 signaling activation. A, After 48 hours of treatment with mock, pSV, pM, pM-FL-A141G, pSV+pM-FL-A141G, pM+pM-FL-A141G, rhMMP-2, pSV+rhMMP-2, and pM+rhMMP-2 as described in Materials and Methods, whole cell lysates were subjected to Western blotting. Blots were representative of at least three independent repetitions and GAPDH probing confirmed equal loading. B, Effect of MMP-2 overexpression by siRNA-insensitive pM-FL-A141G treatment or rhMMP-2 supplementation on pM-inhibited MMP-2/α5β1 integrin binding. Whole cell lysates were (200 µg each) were immunoprecipitated with anti-α5β1 integrin antibody (10 µL each sample) using µMACS™ protein G microbeads and MACS separation columns and immunoprecipitates were subjected to Western blotting. The mock sample immunoprecipitated with non-specific IgG was loaded as the negative control. Input shows whole cell lysate (50 µg) of mock sample served as the positive control. Blots were probed with anti-MMP-2 antibody and representative blots from three independent repetitions were shown. Adjacent bar diagram shows the densitometric analyses of the relative band intensity represented as mean±SE values obtained from three repetitions. The significant differences among various treatment groups were indicated by *at p

    Techniques Used: Over Expression, Activation Assay, Western Blot, Binding Assay, Immunoprecipitation, Magnetic Cell Separation, Negative Control, Positive Control

    16) Product Images from "Identification of the von Hippel-Lindau tumor-suppressor protein as part of an active E3 ubiquitin ligase complex"

    Article Title: Identification of the von Hippel-Lindau tumor-suppressor protein as part of an active E3 ubiquitin ligase complex

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

    doi:

    Identification of P220 and P100 as WT-pVHL specific associated molecules. Immunoprecipitates of HeLa cells expressing Flag-tagged WT, Y98N, R167Q, or vector-transfected HeLa cells with Flag M2 beads were electrophoresed in 5% SDS/PAGE, followed by transfer to nitrocellulose membrane. After proteins were renatured on the membrane, the membrane was incubated with 32 P-labeled VBC complex, followed by autoradiography.
    Figure Legend Snippet: Identification of P220 and P100 as WT-pVHL specific associated molecules. Immunoprecipitates of HeLa cells expressing Flag-tagged WT, Y98N, R167Q, or vector-transfected HeLa cells with Flag M2 beads were electrophoresed in 5% SDS/PAGE, followed by transfer to nitrocellulose membrane. After proteins were renatured on the membrane, the membrane was incubated with 32 P-labeled VBC complex, followed by autoradiography.

    Techniques Used: Expressing, Plasmid Preparation, Transfection, SDS Page, Incubation, Labeling, Autoradiography

    VBC-CUL-2 exhibits the E3 activity together with E2s of UbcH5 family. ( A ) The VBC-CUL-2 complex immunoprecipitated from HA-tagged WT-pVHL expressing 786–0 cells was incubated with recombinant E1, 32 P-labeled ubiquitin, and ATP regeneration system at 37°C for 30 min in the presence of UBC3 (lane 2), UbcH5a (lane 3), UbcH5b (lane 4), UbcH5c (lane 5), UbcH7 (lane 6), E2–25K (lane 7), or E2–20K (lane 8), or in the absence of any E2 (lane 1). ( B ) In vitro ubiquitination reactions were performed as described in Materials and Methods , except for the followings: lane 1 lacks VBC-CUL-2, lane 3 lacks ATP regeneration system, lane 4 lacks UbcH5b, and lane 5 lacks E1. ( C ) Immunoprecipitates with anti-HA agarose beads from HA-tagged WT-pVHL expressing (lane 3), HA-tagged truncated pVHL (amino acids 1–115) expressing (lane 4), or parent 786–0 cells (lane 2) were subjected to the in vitro ubiquitination assay together with anti-HA beads alone (lane 1) in the presence of E1, UbcH5b, 32 P-labeled ubiquitin, and ATP regeneration system at 37°C for 30 min. Reactions were stopped by adding 4× sample buffer and electrophoresed in 10% SDS/PAGE, followed by autoradiography.
    Figure Legend Snippet: VBC-CUL-2 exhibits the E3 activity together with E2s of UbcH5 family. ( A ) The VBC-CUL-2 complex immunoprecipitated from HA-tagged WT-pVHL expressing 786–0 cells was incubated with recombinant E1, 32 P-labeled ubiquitin, and ATP regeneration system at 37°C for 30 min in the presence of UBC3 (lane 2), UbcH5a (lane 3), UbcH5b (lane 4), UbcH5c (lane 5), UbcH7 (lane 6), E2–25K (lane 7), or E2–20K (lane 8), or in the absence of any E2 (lane 1). ( B ) In vitro ubiquitination reactions were performed as described in Materials and Methods , except for the followings: lane 1 lacks VBC-CUL-2, lane 3 lacks ATP regeneration system, lane 4 lacks UbcH5b, and lane 5 lacks E1. ( C ) Immunoprecipitates with anti-HA agarose beads from HA-tagged WT-pVHL expressing (lane 3), HA-tagged truncated pVHL (amino acids 1–115) expressing (lane 4), or parent 786–0 cells (lane 2) were subjected to the in vitro ubiquitination assay together with anti-HA beads alone (lane 1) in the presence of E1, UbcH5b, 32 P-labeled ubiquitin, and ATP regeneration system at 37°C for 30 min. Reactions were stopped by adding 4× sample buffer and electrophoresed in 10% SDS/PAGE, followed by autoradiography.

    Techniques Used: Activity Assay, Immunoprecipitation, Expressing, Incubation, Recombinant, Labeling, In Vitro, Ubiquitin Assay, SDS Page, Autoradiography

    17) Product Images from "Dok-1 Independently Attenuates Ras/Mitogen-Activated Protein Kinase and Src/c-Myc Pathways To Inhibit Platelet-Derived Growth Factor-Induced Mitogenesis"

    Article Title: Dok-1 Independently Attenuates Ras/Mitogen-Activated Protein Kinase and Src/c-Myc Pathways To Inhibit Platelet-Derived Growth Factor-Induced Mitogenesis

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.26.7.2479-2489.2006

    Dok-1 associates with Csk and recruits it to active Src in response to PDGF. (A) Association of Dok-1 WT with Src and Csk in NIH 3T3 cells. NIH 3T3 cells stably transduced with Flag-tagged Dok-1 WT or control vector were serum starved and either left unstimulated (−) or stimulated (+) with PDGF (12.5 ng/ml) for 10 min. Cell extracts were subjected to anti-Flag (α-Flag) immunoprecipitation (IP). A portion (0.2%) of the total cell lysates (TCL) used for immunoprecipitation and immunodepleted lysates and 1% of the final eluates were separated by SDS-PAGE and immunoblotted with anti-Dok-1 (α-Dok-1) Ab (top panel). The remaining eluted immune complexes were analyzed by Western blotting (WB) with anti-Csk (α-Csk; C20) and polyclonal anti-Src Abs (bottom panel). (B) Dok-1 regulates Csk localization. Serum-starved WT and Dok-1 − / − MEFs plated on coverslips were either left untreated (−) or stimulated (+) with PDGF (12.5 ng/ml) for 10 min. Cells were stained with monoclonal anti-Csk Ab. (C) Substitution of Tyr-449 with Phe in Dok-1 abolishes its interaction with Csk. Anti-Flag immunoprecipitates from unstimulated (−) or PDGF (12.5 ng/ml)-stimulated (+) NIH 3T3 cells stably transduced with Flag-tagged Dok-1 WT , Flag-tagged Dok-1 CSK , or control vector were analyzed by immunoblotting with anti-Csk (C20) Ab (top panel). Equivalent amounts of cell lysates used for immunoprecipitation were separated by SDS-PAGE and immunoblotted with anti-Dok-1 Ab to demonstrate equal expression of Dok-1 WT and Dok-1 CSK in NIH 3T3 cells and with anti-p42 MAPK (C14) Ab as a loading control (bottom panel).
    Figure Legend Snippet: Dok-1 associates with Csk and recruits it to active Src in response to PDGF. (A) Association of Dok-1 WT with Src and Csk in NIH 3T3 cells. NIH 3T3 cells stably transduced with Flag-tagged Dok-1 WT or control vector were serum starved and either left unstimulated (−) or stimulated (+) with PDGF (12.5 ng/ml) for 10 min. Cell extracts were subjected to anti-Flag (α-Flag) immunoprecipitation (IP). A portion (0.2%) of the total cell lysates (TCL) used for immunoprecipitation and immunodepleted lysates and 1% of the final eluates were separated by SDS-PAGE and immunoblotted with anti-Dok-1 (α-Dok-1) Ab (top panel). The remaining eluted immune complexes were analyzed by Western blotting (WB) with anti-Csk (α-Csk; C20) and polyclonal anti-Src Abs (bottom panel). (B) Dok-1 regulates Csk localization. Serum-starved WT and Dok-1 − / − MEFs plated on coverslips were either left untreated (−) or stimulated (+) with PDGF (12.5 ng/ml) for 10 min. Cells were stained with monoclonal anti-Csk Ab. (C) Substitution of Tyr-449 with Phe in Dok-1 abolishes its interaction with Csk. Anti-Flag immunoprecipitates from unstimulated (−) or PDGF (12.5 ng/ml)-stimulated (+) NIH 3T3 cells stably transduced with Flag-tagged Dok-1 WT , Flag-tagged Dok-1 CSK , or control vector were analyzed by immunoblotting with anti-Csk (C20) Ab (top panel). Equivalent amounts of cell lysates used for immunoprecipitation were separated by SDS-PAGE and immunoblotted with anti-Dok-1 Ab to demonstrate equal expression of Dok-1 WT and Dok-1 CSK in NIH 3T3 cells and with anti-p42 MAPK (C14) Ab as a loading control (bottom panel).

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

    18) Product Images from "The Schizosaccharomyces pombe dim1+ Gene Interacts with the Anaphase-Promoting Complex or Cyclosome (APC/C) Component lid1+ and Is Required for APC/C Function"

    Article Title: The Schizosaccharomyces pombe dim1+ Gene Interacts with the Anaphase-Promoting Complex or Cyclosome (APC/C) Component lid1+ and Is Required for APC/C Function

    Journal: Molecular and Cellular Biology

    doi:

    lid1p-myc associates with several other proteins in vivo. (A) lid1p-myc is an ∼100-kDa protein. KGY1302 whole-cell lysates (lane 1) or 9E10 immunoprecipitates from KGY1302 lysates (lane 2) were resolved by SDS-PAGE and transferred to a PVDF membrane, and the membrane was probed with the 9E10 antibody. Molecular mass standards (in kilodaltons) are indicated. (B) Wild-type (lanes 1 and 3) or KGY1302 ( lid1 :: lid1-myc ) (lanes 2 and 4) native 35 S-labeled cell lysates were immunoprecipitated with the 9E10 antibody, and immunoprecipitates were resolved by SDS-PAGE. Labeled proteins from two separate experiments for 1 day (lanes 1 and 2) or 7 days (lanes 3 and 4) were visualized by fluorography. Migrations of molecular mass markers (in kilodaltons) are indicated. Bands specific to the KGY1302 immunoprecipitate are indicated (●). An arrowhead indicates the band corresponding to lid1p-myc. (C) Cut9p-HA is an ∼90-kDa protein. A KGY1365 whole-cell lysate (lane 1) or an HA.11 immunoprecipitate (lane 2) was resolved by SDS-PAGE and transferred to a PVDF membrane, and the membrane was probed with the HA.11 antibody.
    Figure Legend Snippet: lid1p-myc associates with several other proteins in vivo. (A) lid1p-myc is an ∼100-kDa protein. KGY1302 whole-cell lysates (lane 1) or 9E10 immunoprecipitates from KGY1302 lysates (lane 2) were resolved by SDS-PAGE and transferred to a PVDF membrane, and the membrane was probed with the 9E10 antibody. Molecular mass standards (in kilodaltons) are indicated. (B) Wild-type (lanes 1 and 3) or KGY1302 ( lid1 :: lid1-myc ) (lanes 2 and 4) native 35 S-labeled cell lysates were immunoprecipitated with the 9E10 antibody, and immunoprecipitates were resolved by SDS-PAGE. Labeled proteins from two separate experiments for 1 day (lanes 1 and 2) or 7 days (lanes 3 and 4) were visualized by fluorography. Migrations of molecular mass markers (in kilodaltons) are indicated. Bands specific to the KGY1302 immunoprecipitate are indicated (●). An arrowhead indicates the band corresponding to lid1p-myc. (C) Cut9p-HA is an ∼90-kDa protein. A KGY1365 whole-cell lysate (lane 1) or an HA.11 immunoprecipitate (lane 2) was resolved by SDS-PAGE and transferred to a PVDF membrane, and the membrane was probed with the HA.11 antibody.

    Techniques Used: In Vivo, SDS Page, Labeling, Immunoprecipitation

    lid1p-myc is a component of the 20S APC/C. (A) lid1p-myc and cut9p-HA cosediment at ∼20S. Fractions were collected from the bottom (fraction 1) of 10 to 30% sucrose gradients and then resolved by SDS-PAGE and immunoblotted with the 9E10 antibody to detect lid1p-myc (a and c) or with 12CA5 to detect cut9-HA (b and d). Panels a and b show identical fractions from lysates prepared from the lid1 :: lid1-myc cut9 :: cut9 ::3xHA strain. Panel c shows fractions from the lid1 :: lid1-myc cut9-665 strain that had been shifted to 36°C for 4 h prior to lysis. Panel d shows fractions from the cut9 :: cut9-HA lid1-6 strain that had been shifted to 36°C for 4 h prior to lysis. Fraction numbers are indicated above the panels. Peak fractions of the 19S sedimentation marker thyroglobulin and the 11.3S sedimentation marker catalase are indicated below the panels. (B) lid1-myc and cut9p-HA coimmunoprecipitate from KGY1366 ( lid1 :: lid1-myc cut9 :: cut9-HA ) whole-cell lysates but not from KGY1302 ( lid1 :: lid1-myc ) or KGY1365 ( cut9 :: cut9-HA ) cell lysates. KGY1302 (lanes 1, 2, 3, and 4), KGY1365 (lanes 5, 6, 7, and 8), or KGY1366 (lanes 9, 10, 11, and 12) native (N) (lanes 1, 3, 5, 7, 9, and 11) or denatured (D) (lanes 2, 4, 6, 8, 10, and 12) cell lysates were immunoprecipitated (IP) with 9E10 (lanes 1, 2, 5, 6, 9, and 10) or HA.11 (lanes 3, 4, 7, 8, 11, and 12) antibodies. Immunoprecipitates were resolved by SDS-PAGE and then subjected to immunoblotting with 9E10 (a) or HA.11 (b) antibodies. (C) lid1p-myc coimmunoprecipitates with cut9p and nuc2p. Polyclonal antibodies to cut9p (lanes 1 and 3) or nuc2p (lanes 2 or 4) were used for immunoprecipitation from wild-type (lanes 1 and 2) or KGY1302 (lanes 3 and 4) cell lysates. The immunoprecipitates were resolved by SDS-PAGE and blotted with 9E10 antibodies. lid1p-myc is indicated with an arrow.
    Figure Legend Snippet: lid1p-myc is a component of the 20S APC/C. (A) lid1p-myc and cut9p-HA cosediment at ∼20S. Fractions were collected from the bottom (fraction 1) of 10 to 30% sucrose gradients and then resolved by SDS-PAGE and immunoblotted with the 9E10 antibody to detect lid1p-myc (a and c) or with 12CA5 to detect cut9-HA (b and d). Panels a and b show identical fractions from lysates prepared from the lid1 :: lid1-myc cut9 :: cut9 ::3xHA strain. Panel c shows fractions from the lid1 :: lid1-myc cut9-665 strain that had been shifted to 36°C for 4 h prior to lysis. Panel d shows fractions from the cut9 :: cut9-HA lid1-6 strain that had been shifted to 36°C for 4 h prior to lysis. Fraction numbers are indicated above the panels. Peak fractions of the 19S sedimentation marker thyroglobulin and the 11.3S sedimentation marker catalase are indicated below the panels. (B) lid1-myc and cut9p-HA coimmunoprecipitate from KGY1366 ( lid1 :: lid1-myc cut9 :: cut9-HA ) whole-cell lysates but not from KGY1302 ( lid1 :: lid1-myc ) or KGY1365 ( cut9 :: cut9-HA ) cell lysates. KGY1302 (lanes 1, 2, 3, and 4), KGY1365 (lanes 5, 6, 7, and 8), or KGY1366 (lanes 9, 10, 11, and 12) native (N) (lanes 1, 3, 5, 7, 9, and 11) or denatured (D) (lanes 2, 4, 6, 8, 10, and 12) cell lysates were immunoprecipitated (IP) with 9E10 (lanes 1, 2, 5, 6, 9, and 10) or HA.11 (lanes 3, 4, 7, 8, 11, and 12) antibodies. Immunoprecipitates were resolved by SDS-PAGE and then subjected to immunoblotting with 9E10 (a) or HA.11 (b) antibodies. (C) lid1p-myc coimmunoprecipitates with cut9p and nuc2p. Polyclonal antibodies to cut9p (lanes 1 and 3) or nuc2p (lanes 2 or 4) were used for immunoprecipitation from wild-type (lanes 1 and 2) or KGY1302 (lanes 3 and 4) cell lysates. The immunoprecipitates were resolved by SDS-PAGE and blotted with 9E10 antibodies. lid1p-myc is indicated with an arrow.

    Techniques Used: SDS Page, Lysis, Sedimentation, Marker, Immunoprecipitation

    19) Product Images from "Cyclin I is involved in the regulation of cell cycle progression"

    Article Title: Cyclin I is involved in the regulation of cell cycle progression

    Journal: Cell Cycle

    doi: 10.4161/cc.25623

    Figure 2. Cyclin I is degraded via the ubiquitin-proteasome pathway. ( A and B ) Increasing level of Cyclin I by proteasome inhibition. HEK293T cells were transfected with pDsRed-Cyclin I. After incubation for 16 h, cells were treated with or without 10 μg/ml lactacystin for 24 h. Cells were observed with fluorescence microscope ( A ) or the lysates were fractionated by SDS-PAGE and immunoblotted with antibodies as indicated ( B ). Bars, 50 μm. ( C ) Ubiquitination of Cyclin I. HEK293T cells were transfected with pTB701-Flag-Cyclin I-Wt, pTB701-Flag-Cyclin I-ΔPEST, and pCGN-HA-ubiquitin as indicated. After incubation for 24 h, cells were treated with 10 μg/ml lactacystin for 6 h, and the lysates were immunoprecipitated with anti-Flag M2 agarose beads. The input lysates and the immunoprecipitates were fractionated by SDS-PAGE and immunoblotted with antibodies as indicated.
    Figure Legend Snippet: Figure 2. Cyclin I is degraded via the ubiquitin-proteasome pathway. ( A and B ) Increasing level of Cyclin I by proteasome inhibition. HEK293T cells were transfected with pDsRed-Cyclin I. After incubation for 16 h, cells were treated with or without 10 μg/ml lactacystin for 24 h. Cells were observed with fluorescence microscope ( A ) or the lysates were fractionated by SDS-PAGE and immunoblotted with antibodies as indicated ( B ). Bars, 50 μm. ( C ) Ubiquitination of Cyclin I. HEK293T cells were transfected with pTB701-Flag-Cyclin I-Wt, pTB701-Flag-Cyclin I-ΔPEST, and pCGN-HA-ubiquitin as indicated. After incubation for 24 h, cells were treated with 10 μg/ml lactacystin for 6 h, and the lysates were immunoprecipitated with anti-Flag M2 agarose beads. The input lysates and the immunoprecipitates were fractionated by SDS-PAGE and immunoblotted with antibodies as indicated.

    Techniques Used: Inhibition, Transfection, Incubation, Fluorescence, Microscopy, SDS Page, Immunoprecipitation

    Figure 3. Cyclin I associates with Cdk5 in vivo. ( A and B ) Coprecipitation of Cyclin I with Cdk5 in transiently overexpressed HEK293T cells. Lysates from HEK293T cells transfected with pTB701-Flag-Cyclin I and pcDNA3-HA-Cdk5 were immunoprecipitated with anti-Flag agarose beads ( A ) or anti-HA antibody ( B ). The input lysates and the immunoprecipitates were fractionated by SDS-PAGE and immunoblotted with antibodies as indicated. ( C ) Colocalization of Cyclin I and Cdk5 in transiently overexpressed HeLa cells. HeLa cells were transfected with pTB701-Flag-Cyclin I and pcDNA3-HA-Cdk5. After fixation and permeabilization, cells were incubated with anti-Cyclin I antibody and anti-HA antibody, followed by staining nuclei with Hoechst 33258. Bars, 10 μm.
    Figure Legend Snippet: Figure 3. Cyclin I associates with Cdk5 in vivo. ( A and B ) Coprecipitation of Cyclin I with Cdk5 in transiently overexpressed HEK293T cells. Lysates from HEK293T cells transfected with pTB701-Flag-Cyclin I and pcDNA3-HA-Cdk5 were immunoprecipitated with anti-Flag agarose beads ( A ) or anti-HA antibody ( B ). The input lysates and the immunoprecipitates were fractionated by SDS-PAGE and immunoblotted with antibodies as indicated. ( C ) Colocalization of Cyclin I and Cdk5 in transiently overexpressed HeLa cells. HeLa cells were transfected with pTB701-Flag-Cyclin I and pcDNA3-HA-Cdk5. After fixation and permeabilization, cells were incubated with anti-Cyclin I antibody and anti-HA antibody, followed by staining nuclei with Hoechst 33258. Bars, 10 μm.

    Techniques Used: In Vivo, Transfection, Immunoprecipitation, SDS Page, Incubation, Staining

    20) Product Images from "Heat shock protein 90 regulates the expression of Wilms tumor 1 protein in myeloid leukemias"

    Article Title: Heat shock protein 90 regulates the expression of Wilms tumor 1 protein in myeloid leukemias

    Journal: Blood

    doi: 10.1182/blood-2009-10-247239

    Direct interaction between WT1 and Hsp90 . (A) Equal amounts of K562 protein extracts were immunoprecipitated (IP) with agarose-conjugated mouse immunoglobulin G (IgG) or anti-Hsp90 antibodies, and immunoprecipitates were subjected to SDS-PAGE and immunoblotted (IB) for WT1 (top panel) and Hsp90 (bottom panel). Input represents ∼5% of the total protein extract used for immunoprecipitation. (B) Subcellular colocalization of WT1 and Hsp90. K562 cells were stained with DAPI (blue, nuclear stain) and antibodies to WT1 (green) or Hsp90 (red), and confocal images were acquired at 100× magnification. (C) GST pull-down assay. In vitro–translated and 35 S-methionine–labeled full-length Hsp90 was incubated with GST or GST-WT1 protein immobilized on glutathione-sepharose beads, and bound WT1 was detected by fluorography (top panel). 20% of the in vitro–translated protein was used for pull-downs. The bottom panel shows the purity of GST-fused proteins on a Coomassie blue-stained SDS-PAGE gel. (D) Dose-dependent binding of Hsp90 to WT1. Increasing amounts of 35 S-methionine–labeled Hsp90 were added to GST-WT1, and binding was analyzed by autoradiography.
    Figure Legend Snippet: Direct interaction between WT1 and Hsp90 . (A) Equal amounts of K562 protein extracts were immunoprecipitated (IP) with agarose-conjugated mouse immunoglobulin G (IgG) or anti-Hsp90 antibodies, and immunoprecipitates were subjected to SDS-PAGE and immunoblotted (IB) for WT1 (top panel) and Hsp90 (bottom panel). Input represents ∼5% of the total protein extract used for immunoprecipitation. (B) Subcellular colocalization of WT1 and Hsp90. K562 cells were stained with DAPI (blue, nuclear stain) and antibodies to WT1 (green) or Hsp90 (red), and confocal images were acquired at 100× magnification. (C) GST pull-down assay. In vitro–translated and 35 S-methionine–labeled full-length Hsp90 was incubated with GST or GST-WT1 protein immobilized on glutathione-sepharose beads, and bound WT1 was detected by fluorography (top panel). 20% of the in vitro–translated protein was used for pull-downs. The bottom panel shows the purity of GST-fused proteins on a Coomassie blue-stained SDS-PAGE gel. (D) Dose-dependent binding of Hsp90 to WT1. Increasing amounts of 35 S-methionine–labeled Hsp90 were added to GST-WT1, and binding was analyzed by autoradiography.

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

    21) Product Images from "A Disalicylic Acid-Furanyl Derivative Inhibits Ephrin Binding to a Subset of Eph Receptors"

    Article Title: A Disalicylic Acid-Furanyl Derivative Inhibits Ephrin Binding to a Subset of Eph Receptors

    Journal: Chemical biology & drug design

    doi: 10.1111/j.1747-0285.2011.01199.x

    Compound 76D10 inhibits EphA4 and EphA2 activation and cell retraction after ephrin stimulation. (A) Cells pretreated with the indicated concentrations of 76D10 for 15 min were stimulated for 20 min with ephrin Fc (+) or Fc (−) as a control in the continued presence of the compound. COS cells were stimulated with 0.2 μg/mL ephrin-A1 or 0.8 μg/mL ephrin-B2 Fc and used to immunoprecipitate EphA2 and EphB2, while HT22 neuronal cells were stimulated with 0.2 μg/mL ephrin-A1 and used to immunoprecipitate EphA4. Eph immunoprecipitates were probed with anti-phosphotyrosine antibody (PTyr) and reprobed for the Eph receptor immunoprecipitated. (B) PC3 cells pretreated for 15 min with the indicated concentrations of 76D10 were stimulated with 0.2 μg/mL ephrin-A1 Fc (+) or Fc as a control (−) for 20 min in the continued presence of the compound. The histogram shows the average level of phosphorylated EphA2 normalized to the total amount of receptor in the cell lysates, both measured in ELISA assays. Error bars represent standard errors from 4–10 measurements. The levels of EphA2 phosphorylation in cells treated with ephrin-A1 Fc and compound were compared to those in cells treated only with ephrin-A1 Fc by one-way ANOVA and Dunnett’s post test. ***P
    Figure Legend Snippet: Compound 76D10 inhibits EphA4 and EphA2 activation and cell retraction after ephrin stimulation. (A) Cells pretreated with the indicated concentrations of 76D10 for 15 min were stimulated for 20 min with ephrin Fc (+) or Fc (−) as a control in the continued presence of the compound. COS cells were stimulated with 0.2 μg/mL ephrin-A1 or 0.8 μg/mL ephrin-B2 Fc and used to immunoprecipitate EphA2 and EphB2, while HT22 neuronal cells were stimulated with 0.2 μg/mL ephrin-A1 and used to immunoprecipitate EphA4. Eph immunoprecipitates were probed with anti-phosphotyrosine antibody (PTyr) and reprobed for the Eph receptor immunoprecipitated. (B) PC3 cells pretreated for 15 min with the indicated concentrations of 76D10 were stimulated with 0.2 μg/mL ephrin-A1 Fc (+) or Fc as a control (−) for 20 min in the continued presence of the compound. The histogram shows the average level of phosphorylated EphA2 normalized to the total amount of receptor in the cell lysates, both measured in ELISA assays. Error bars represent standard errors from 4–10 measurements. The levels of EphA2 phosphorylation in cells treated with ephrin-A1 Fc and compound were compared to those in cells treated only with ephrin-A1 Fc by one-way ANOVA and Dunnett’s post test. ***P

    Techniques Used: Activation Assay, Immunoprecipitation, Enzyme-linked Immunosorbent Assay

    22) Product Images from "CDK1-Cyclin B1 Activates RNMT, Coordinating mRNA Cap Methylation with G1 Phase Transcription"

    Article Title: CDK1-Cyclin B1 Activates RNMT, Coordinating mRNA Cap Methylation with G1 Phase Transcription

    Journal: Molecular Cell

    doi: 10.1016/j.molcel.2016.02.008

    RNMT Phosphorylation Reduces Inhibition by KPNA2 (A) HeLa cells expressing HA-RNMT WT, T77A, T77D, or vector control (C) were transfected with pcDNA5 Myc-KPNA2 or vector control (C). 9E10 antibody was used to immunoprecipitate Myc-KPNA2, and WBs were performed to detect HA-RNMT, RNMT, and Myc-KPNA2. (B) KPNA2 IP was performed on HeLa cells expressing HA-RNMT WT, T77A, T77D, or a vector control using anti-KPNA2 antibody, and KPNA2 and RNMT were detected by WB. (C) HeLa cells were transfected with pcDNA5 Myc-KPNA2. Cells were released from double thymidine block for 2 hr (S) or 8 hr (G2/M), including incubation with 9 μM RO-3306 for 15 min (G2/M RO). Asynchronous cells transfected with the vector control were used as a control. 9E10 antibody was used to immunoprecipitate Myc-KPNA2, and WBs were performed to detect RNMT and KPNA2. (D) HeLa cells were treated as in (C), except that G2/M cells were also treated with 50 μM roscovitine (G2/M Rosc) for 15 min. Anti-KPNA2 antibodies were used to immunoprecipitate KPNA2 from cell extracts using anti-Tubulin antibodies as a control. WBs were performed to detect KPNA2, RNMT, and Tubulin in extracts and immunoprecipitates. (E) A cap methyltransferase assay was performed using 40 nM recombinant RNMT and titration of recombinant KPNA2 or BSA. Activity is reported relative to the RNMT control. (F) Recombinant RNMT and RAM were incubated with recombinant GST-KPNA2 WT, 1–455, 72–529, or GST alone, and complexes were affinity-purified with glutathione-Sepharose. Inputs and eluates were resolved by SDS-PAGE and Coomassie blue-stained, and RAM was detected by WB. (G) HeLa cells were transfected with pcDNA5 Myc-KPNA2 WT, 1–455, 72–529, or vector control (C). 9E10 antibody was used to immunoprecipitate Myc-KPNA2, and WB was performed to detect Myc-KPNA2, KPNA2, RNMT, and Tubulin. (Note that the KPNA2 antibody raised against the N terminus does not recognize KPNA2 72–529.) (H) A cap methyltransferase assay was performed using recombinant RNMT-RAM and recombinant KPNA2 WT, 1–455, 72–529, or GST alone. The charts depict the average and SD of four experiments. ∗∗ p
    Figure Legend Snippet: RNMT Phosphorylation Reduces Inhibition by KPNA2 (A) HeLa cells expressing HA-RNMT WT, T77A, T77D, or vector control (C) were transfected with pcDNA5 Myc-KPNA2 or vector control (C). 9E10 antibody was used to immunoprecipitate Myc-KPNA2, and WBs were performed to detect HA-RNMT, RNMT, and Myc-KPNA2. (B) KPNA2 IP was performed on HeLa cells expressing HA-RNMT WT, T77A, T77D, or a vector control using anti-KPNA2 antibody, and KPNA2 and RNMT were detected by WB. (C) HeLa cells were transfected with pcDNA5 Myc-KPNA2. Cells were released from double thymidine block for 2 hr (S) or 8 hr (G2/M), including incubation with 9 μM RO-3306 for 15 min (G2/M RO). Asynchronous cells transfected with the vector control were used as a control. 9E10 antibody was used to immunoprecipitate Myc-KPNA2, and WBs were performed to detect RNMT and KPNA2. (D) HeLa cells were treated as in (C), except that G2/M cells were also treated with 50 μM roscovitine (G2/M Rosc) for 15 min. Anti-KPNA2 antibodies were used to immunoprecipitate KPNA2 from cell extracts using anti-Tubulin antibodies as a control. WBs were performed to detect KPNA2, RNMT, and Tubulin in extracts and immunoprecipitates. (E) A cap methyltransferase assay was performed using 40 nM recombinant RNMT and titration of recombinant KPNA2 or BSA. Activity is reported relative to the RNMT control. (F) Recombinant RNMT and RAM were incubated with recombinant GST-KPNA2 WT, 1–455, 72–529, or GST alone, and complexes were affinity-purified with glutathione-Sepharose. Inputs and eluates were resolved by SDS-PAGE and Coomassie blue-stained, and RAM was detected by WB. (G) HeLa cells were transfected with pcDNA5 Myc-KPNA2 WT, 1–455, 72–529, or vector control (C). 9E10 antibody was used to immunoprecipitate Myc-KPNA2, and WB was performed to detect Myc-KPNA2, KPNA2, RNMT, and Tubulin. (Note that the KPNA2 antibody raised against the N terminus does not recognize KPNA2 72–529.) (H) A cap methyltransferase assay was performed using recombinant RNMT-RAM and recombinant KPNA2 WT, 1–455, 72–529, or GST alone. The charts depict the average and SD of four experiments. ∗∗ p

    Techniques Used: Inhibition, Expressing, Plasmid Preparation, Transfection, Western Blot, Blocking Assay, Incubation, Recombinant, Titration, Activity Assay, Affinity Purification, SDS Page, Staining

    RNMT T77 Is Phosphorylated during G2/M Phase (A) HeLa cells expressing HA-RNMT were synchronized by double thymidine or thymidine-nocodazole block and released as indicated. In these cells, asynchronous cells (A) and asynchronous cells expressing HA-RNMT T77A (77A), WBs were performed to detect pT77 and total RNMT in HA-RNMT immunoprecipitates and cell cycle markers indicated in cell extracts. Above the blots, a chart depicts the average pT77/RNMT signal and SD for three independent experiments, calculated from WB using ImageJ software. Flow cytometry cell cycle analysis and phase percentage are reported below the blots. (B) HeLa cells were synchronized, and endogenous pT77 and total RNMT were detected in RNMT immunoprecipitates.
    Figure Legend Snippet: RNMT T77 Is Phosphorylated during G2/M Phase (A) HeLa cells expressing HA-RNMT were synchronized by double thymidine or thymidine-nocodazole block and released as indicated. In these cells, asynchronous cells (A) and asynchronous cells expressing HA-RNMT T77A (77A), WBs were performed to detect pT77 and total RNMT in HA-RNMT immunoprecipitates and cell cycle markers indicated in cell extracts. Above the blots, a chart depicts the average pT77/RNMT signal and SD for three independent experiments, calculated from WB using ImageJ software. Flow cytometry cell cycle analysis and phase percentage are reported below the blots. (B) HeLa cells were synchronized, and endogenous pT77 and total RNMT were detected in RNMT immunoprecipitates.

    Techniques Used: Expressing, Blocking Assay, Western Blot, Software, Flow Cytometry, Cytometry, Cell Cycle Assay

    CDK1-Cyclin B1 Phosphorylates and Activates RNMT HeLa cells were transfected with CDK1, CDK3, cyclin B1, and cyclin E1 siRNA. (A and B) After 48 hr, (A) pT77 and total RNMT levels were analyzed in RNMT immunoprecipitates, and (B) CDK and cyclin levels were analyzed in cell extracts by WB. c, control. (C) Recombinant His-RNMT (WT or T77A)-GST-RAM was incubated with activated CDK1-cyclin B1 and 32 P-ATP for 60 min and resolved by SDS-PAGE. Labeled bands were visualized by phosphoimaging. pT77 and total RNMT were visualized by WB and Coomassie stain. (D) To assess enzyme kinetics, the CDK1-cyclin B1 kinase reaction was performed using a titration of RNMT or histone H1 over a time course ( Figure S1 ). The charts depict reaction velocities for phosphorylation of RNMT-RAM or histone H1. Error bars represent SD for reaction velocity at each substrate concentration. k cat and K M values were calculated using an allosteric sigmoidal curve fit. (E) As in (C), except a time course experiment was performed. Quantitation of the ATP:RNMT incorporation ratio is reported above. (F) His-RNMT-GST-RAM was incubated with activated CDK1-cyclin B1 and ATP for 10 min as in (E) and then utilized in the cap methyltransferase assay. rxn, reaction. (G) His-RNMT WT or 77A-GST-RAM were incubated in the presence and absence of ATP and CDK1-cyclin B1 for 10 min as above and then utilized in the cap methyltransferase assay. Average and SD for three independent experiments is depicted. ∗ p
    Figure Legend Snippet: CDK1-Cyclin B1 Phosphorylates and Activates RNMT HeLa cells were transfected with CDK1, CDK3, cyclin B1, and cyclin E1 siRNA. (A and B) After 48 hr, (A) pT77 and total RNMT levels were analyzed in RNMT immunoprecipitates, and (B) CDK and cyclin levels were analyzed in cell extracts by WB. c, control. (C) Recombinant His-RNMT (WT or T77A)-GST-RAM was incubated with activated CDK1-cyclin B1 and 32 P-ATP for 60 min and resolved by SDS-PAGE. Labeled bands were visualized by phosphoimaging. pT77 and total RNMT were visualized by WB and Coomassie stain. (D) To assess enzyme kinetics, the CDK1-cyclin B1 kinase reaction was performed using a titration of RNMT or histone H1 over a time course ( Figure S1 ). The charts depict reaction velocities for phosphorylation of RNMT-RAM or histone H1. Error bars represent SD for reaction velocity at each substrate concentration. k cat and K M values were calculated using an allosteric sigmoidal curve fit. (E) As in (C), except a time course experiment was performed. Quantitation of the ATP:RNMT incorporation ratio is reported above. (F) His-RNMT-GST-RAM was incubated with activated CDK1-cyclin B1 and ATP for 10 min as in (E) and then utilized in the cap methyltransferase assay. rxn, reaction. (G) His-RNMT WT or 77A-GST-RAM were incubated in the presence and absence of ATP and CDK1-cyclin B1 for 10 min as above and then utilized in the cap methyltransferase assay. Average and SD for three independent experiments is depicted. ∗ p

    Techniques Used: Transfection, Western Blot, Recombinant, Incubation, SDS Page, Labeling, Staining, Titration, Concentration Assay, Quantitation Assay

    RNMT Is Phosphorylated on T77 (A) HA-RNMT-expressing 293 cells were cultured with 32 P orthophosphate, HA-RNMT-immunoprecipitated via an HA tag, and resolved by SDS-PAGE, and proteins were detected by phosphoimaging (phos) and western blot (WB). vec, vector. (B) Cells were transfected with RNMT or control (con) siRNA for 48 hr and then cultured with 32 P orthophosphate. Endogenous RNMT IP was performed using anti-RNMT antibodies, and protein was detected. Anti-GST antibodies were used as a control. (C) A phosphorylated RNMT peptide was identified in HA-RNMT by LC-MS/MS precursor ion scan. Potential phosphorylated amino acids are underlined. Mascot score (MS) and expected value (Exp) are indicated. (D) HeLa cells expressing HA-RNMT WT, T77A-S79A, T77A, and S79A were cultured with 32 P orthophosphate and analyzed as in (A). Phospho-RNMT in two independent experiments was quantified relative to RNMT WT. Average and SD are depicted. (E) WBs were performed to detect pT77 and total RNMT in HA-RNMT WT and T77A immunoprecipitates with (+PI) and without (−PI) phosphatase inhibitors. (F) Endogenous pT77 and total RNMT were detected in RNMT immunoprecipitates from HeLa cells. (G) CDK and cyclin binding motifs in RNMT. (H–L) HeLa cells expressing HA-RNMT were incubated with (H) 50 μM roscovitine, (I) a titration of roscovitine, (J) 9 μM RO-3306, (K) 1 μM flavopiridol, or (L) 50μM DRB for the times indicated. pT77 and total RNMT were detected in HA-RNMT immunoprecipitates.
    Figure Legend Snippet: RNMT Is Phosphorylated on T77 (A) HA-RNMT-expressing 293 cells were cultured with 32 P orthophosphate, HA-RNMT-immunoprecipitated via an HA tag, and resolved by SDS-PAGE, and proteins were detected by phosphoimaging (phos) and western blot (WB). vec, vector. (B) Cells were transfected with RNMT or control (con) siRNA for 48 hr and then cultured with 32 P orthophosphate. Endogenous RNMT IP was performed using anti-RNMT antibodies, and protein was detected. Anti-GST antibodies were used as a control. (C) A phosphorylated RNMT peptide was identified in HA-RNMT by LC-MS/MS precursor ion scan. Potential phosphorylated amino acids are underlined. Mascot score (MS) and expected value (Exp) are indicated. (D) HeLa cells expressing HA-RNMT WT, T77A-S79A, T77A, and S79A were cultured with 32 P orthophosphate and analyzed as in (A). Phospho-RNMT in two independent experiments was quantified relative to RNMT WT. Average and SD are depicted. (E) WBs were performed to detect pT77 and total RNMT in HA-RNMT WT and T77A immunoprecipitates with (+PI) and without (−PI) phosphatase inhibitors. (F) Endogenous pT77 and total RNMT were detected in RNMT immunoprecipitates from HeLa cells. (G) CDK and cyclin binding motifs in RNMT. (H–L) HeLa cells expressing HA-RNMT were incubated with (H) 50 μM roscovitine, (I) a titration of roscovitine, (J) 9 μM RO-3306, (K) 1 μM flavopiridol, or (L) 50μM DRB for the times indicated. pT77 and total RNMT were detected in HA-RNMT immunoprecipitates.

    Techniques Used: Expressing, Cell Culture, Immunoprecipitation, SDS Page, Western Blot, Plasmid Preparation, Transfection, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Binding Assay, Incubation, Titration

    RNMT Phosphorylation Is Required for Gene Expression (A) HeLa cells synchronized by thymidine-nocodazole block were released into G1 phase for the hours indicated. pT77 and total RNMT were detected in RNMT immunoprecipitates by WB. Relative cap methyltransferase activity of the same cell extracts was determined. A sample result is presented, indicating migration of substrate, GpppG, and product, m7GpppG, on TLC. Average and SD are presented in the chart for three biologically independent experiments. (B) As in (A), except cells were treated with 50 μM roscovitine for 30 min prior to analyses. v, vector. (C) Label-free proteomic analysis of HeLa cells expressing HA-RNMT WT and T77A was performed on three independent samples. Data were analyzed by Maxquant for significance, and identified proteins are represented by volcano plot depicting the average expression ratio in cells expressing HA-RNMT 77A versus WT. (D) REVIGO rationalization of GO term analysis of proteins repressed in cells expressing RNMT T77A in comparison with WT. Axes represent semantic space used to group GO terms of related biological processes. The color and size of each bubble indicates the p value for each GO term versus the human proteome. The bubble size increases and color changes from red to blue with increasing p value. The proximity of the bubbles reflects the relatedness of GO terms. (E) BOP-1, TOMM70A, DDX18, and Skp2 were detected by WB in extracts of cells expressing HA-RNMT WT, T77A, and vector control. For three independent preparations of cells expressing HA-RNMT WT and 77A, the charts indicate the average label-free quantitation (LFQ) and SD of proteins detected in the mass spectrometry analysis. (F) RNA was immunoprecipitated from the same cells using an anti-m7G antibody or control, and RT-PCR was used to detect m7G-capped transcripts relative to total transcripts. ∗ p > 0.05, ∗∗ p > 0.001, t test. See also Tables S1 , S2 , and S3 .
    Figure Legend Snippet: RNMT Phosphorylation Is Required for Gene Expression (A) HeLa cells synchronized by thymidine-nocodazole block were released into G1 phase for the hours indicated. pT77 and total RNMT were detected in RNMT immunoprecipitates by WB. Relative cap methyltransferase activity of the same cell extracts was determined. A sample result is presented, indicating migration of substrate, GpppG, and product, m7GpppG, on TLC. Average and SD are presented in the chart for three biologically independent experiments. (B) As in (A), except cells were treated with 50 μM roscovitine for 30 min prior to analyses. v, vector. (C) Label-free proteomic analysis of HeLa cells expressing HA-RNMT WT and T77A was performed on three independent samples. Data were analyzed by Maxquant for significance, and identified proteins are represented by volcano plot depicting the average expression ratio in cells expressing HA-RNMT 77A versus WT. (D) REVIGO rationalization of GO term analysis of proteins repressed in cells expressing RNMT T77A in comparison with WT. Axes represent semantic space used to group GO terms of related biological processes. The color and size of each bubble indicates the p value for each GO term versus the human proteome. The bubble size increases and color changes from red to blue with increasing p value. The proximity of the bubbles reflects the relatedness of GO terms. (E) BOP-1, TOMM70A, DDX18, and Skp2 were detected by WB in extracts of cells expressing HA-RNMT WT, T77A, and vector control. For three independent preparations of cells expressing HA-RNMT WT and 77A, the charts indicate the average label-free quantitation (LFQ) and SD of proteins detected in the mass spectrometry analysis. (F) RNA was immunoprecipitated from the same cells using an anti-m7G antibody or control, and RT-PCR was used to detect m7G-capped transcripts relative to total transcripts. ∗ p > 0.05, ∗∗ p > 0.001, t test. See also Tables S1 , S2 , and S3 .

    Techniques Used: Expressing, Blocking Assay, Western Blot, Activity Assay, Migration, Thin Layer Chromatography, Plasmid Preparation, Quantitation Assay, Mass Spectrometry, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

    23) Product Images from "A truncated isoform of the PP2A B56 subunit promotes cell motility through paxillin phosphorylation"

    Article Title: A truncated isoform of the PP2A B56 subunit promotes cell motility through paxillin phosphorylation

    Journal: The EMBO Journal

    doi: 10.1093/emboj/19.4.562

    Fig. 6. Co-localization of Δγ1 with paxillin. ( A ) Co-immunoprecipitation of paxillin and PP2A B56γ isoforms by PP2A C subunit Ab. Cells were lysed 30 min after non-adherent cell cultivation and immunoprecipitated with PP2A C subunit Ab. The blot was probed with a mixture of paxillin (mAb) and PP2A C antibodies (upper panel), or B56γ Ab alone (lower panel). ( B ) Cells were lysed 30 min after being plated on FN, and immunoprecipitated with paxillin mAb. The immunoprecipitates and whole-cell lysate (50 μg) of 3T3 Δγ1 -1 cells were blotted and probed with anti-B56γ Ab. The positions of the B56γ isoforms, γ3, γ2, γ1 and Δγ1, are indicated on the left of the blots in (A) and (B). NC indicates a representative result of negative controls in (A) and (B). Arrows in (A) and (B) indicate the position of the IgG heavy chain. ( C ) Subcellular localization of HA-tagged B56γ3, γ2, γ1 and Δγ1 proteins. COS-7 cells were transiently transfected with HA-tagged full length B56γ3 (COS-7 HA-γ3 ), γ2 (COS-7 HA-γ2 ), γ1 (COS-7 HA-γ1 ) or Δγ1 (COS-7 HA-Δγ1 ) vector. After transfection, cells were replaced on chamber slides, reacted with anti-HA and paxillin polyclonal Ab, and stained with Cy2 (green; upper row) and Cy3 (red; center row), respectively. Cy2 and Cy3 images were merged into one (lower row).
    Figure Legend Snippet: Fig. 6. Co-localization of Δγ1 with paxillin. ( A ) Co-immunoprecipitation of paxillin and PP2A B56γ isoforms by PP2A C subunit Ab. Cells were lysed 30 min after non-adherent cell cultivation and immunoprecipitated with PP2A C subunit Ab. The blot was probed with a mixture of paxillin (mAb) and PP2A C antibodies (upper panel), or B56γ Ab alone (lower panel). ( B ) Cells were lysed 30 min after being plated on FN, and immunoprecipitated with paxillin mAb. The immunoprecipitates and whole-cell lysate (50 μg) of 3T3 Δγ1 -1 cells were blotted and probed with anti-B56γ Ab. The positions of the B56γ isoforms, γ3, γ2, γ1 and Δγ1, are indicated on the left of the blots in (A) and (B). NC indicates a representative result of negative controls in (A) and (B). Arrows in (A) and (B) indicate the position of the IgG heavy chain. ( C ) Subcellular localization of HA-tagged B56γ3, γ2, γ1 and Δγ1 proteins. COS-7 cells were transiently transfected with HA-tagged full length B56γ3 (COS-7 HA-γ3 ), γ2 (COS-7 HA-γ2 ), γ1 (COS-7 HA-γ1 ) or Δγ1 (COS-7 HA-Δγ1 ) vector. After transfection, cells were replaced on chamber slides, reacted with anti-HA and paxillin polyclonal Ab, and stained with Cy2 (green; upper row) and Cy3 (red; center row), respectively. Cy2 and Cy3 images were merged into one (lower row).

    Techniques Used: Immunoprecipitation, Transfection, Plasmid Preparation, Staining

    Fig. 5. Enhanced paxillin phosphorylation on serine residues in 3T3 Δγ1 cells. ( A ) Cells were lysed at 30 min after non-adherent culture (suspension), and 30 (FN 30min) and 90 (FN 90min) min after plating on FN. Immunoprecipitates with paxillin mAb (upper and center panels) and whole-cell lysates (lower panels) were separated by SDS–PAGE. The blots of immunoprecipitates were probed with a mixture of antibodies against FAK, paxillin (mAb) and PP2A C subunit (upper panel), or anti-phosphotyrosine Ab alone (4G10, center panel). Protein expression in the whole-cell lysates was also examined with antibodies against PP2A C subunit, FAK and vinculin (lower panels). NC indicates a representative result of negative controls. White and black arrowheads beside paxillin indicate phosphorylated and unphosphorylated paxillin, respectively. The arrow on the right indicates the position of the IgG heavy chain. ( B ) Phosphoamino acid analysis of 3T3 γ1 and 3T3 Δγ1 -1 cells. Immunoprecipitated paxillin with a radioactivity of 500 c.p.m. was hydrolyzed and loaded onto a thin-layer chromatography plate. Co-migration of ninhydrin-stained phosphoamino acid standards is indicated on the right. P i indicates the position of free 32 P-labeled inorganic phosphate. Lower spots are partial hydrolysis products (part.). ( C ) Quantification of phosphorylation on serine, threonine and tyrosine residues. Signal intensity for each residue, P i or partial hydrolysis products in (B) was quantified and expressed as a percentage of the total intensity.
    Figure Legend Snippet: Fig. 5. Enhanced paxillin phosphorylation on serine residues in 3T3 Δγ1 cells. ( A ) Cells were lysed at 30 min after non-adherent culture (suspension), and 30 (FN 30min) and 90 (FN 90min) min after plating on FN. Immunoprecipitates with paxillin mAb (upper and center panels) and whole-cell lysates (lower panels) were separated by SDS–PAGE. The blots of immunoprecipitates were probed with a mixture of antibodies against FAK, paxillin (mAb) and PP2A C subunit (upper panel), or anti-phosphotyrosine Ab alone (4G10, center panel). Protein expression in the whole-cell lysates was also examined with antibodies against PP2A C subunit, FAK and vinculin (lower panels). NC indicates a representative result of negative controls. White and black arrowheads beside paxillin indicate phosphorylated and unphosphorylated paxillin, respectively. The arrow on the right indicates the position of the IgG heavy chain. ( B ) Phosphoamino acid analysis of 3T3 γ1 and 3T3 Δγ1 -1 cells. Immunoprecipitated paxillin with a radioactivity of 500 c.p.m. was hydrolyzed and loaded onto a thin-layer chromatography plate. Co-migration of ninhydrin-stained phosphoamino acid standards is indicated on the right. P i indicates the position of free 32 P-labeled inorganic phosphate. Lower spots are partial hydrolysis products (part.). ( C ) Quantification of phosphorylation on serine, threonine and tyrosine residues. Signal intensity for each residue, P i or partial hydrolysis products in (B) was quantified and expressed as a percentage of the total intensity.

    Techniques Used: SDS Page, Expressing, Phosphoamino Acid Analysis, Immunoprecipitation, Radioactivity, Thin Layer Chromatography, Migration, Staining, Labeling

    24) Product Images from "Tail-anchored PEX26 targets peroxisomes via a PEX19-dependent and TRC40-independent class I pathway"

    Article Title: Tail-anchored PEX26 targets peroxisomes via a PEX19-dependent and TRC40-independent class I pathway

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201211077

    Capture by TRC40 hinders the potential of PEX19 to interact with Sec61β-R and VAMP2-R. (A) HeLa cells each transiently expressing EGFP-OMP25 (a), EGFP–Cyt b 5 (b), EGFP-Sec61β (c), and EGFP-VAPM2 (d) proteins (WT, lanes 1 and 2; R, lanes 3 and 4) in combination with Myc-PEX19 or FLAG-PEX19 were lysed and subjected to immunoprecipitation with anti-FLAG agarose beads. Immunoprecipitates and input (10%) were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. Note that endogenous PEX19 is not visible at this exposure of the blot. (B and C) FLAG-tagged proteins indicated at the top were synthesized in RRL in the absence (B) or presence (C) of RRL-synthesized HA-PEX19 and then immunoprecipitated as in Fig. 5 A . Immunoprecipitates and input (10%) were analyzed by immunoblotting using the indicated antibodies. Solid and open arrowheads indicate unmodified and farnesylated HA-PEX19, respectively. Black line indicates that intervening lanes have been spliced out. IB, immunoblot; IP, immunoprecipitation.
    Figure Legend Snippet: Capture by TRC40 hinders the potential of PEX19 to interact with Sec61β-R and VAMP2-R. (A) HeLa cells each transiently expressing EGFP-OMP25 (a), EGFP–Cyt b 5 (b), EGFP-Sec61β (c), and EGFP-VAPM2 (d) proteins (WT, lanes 1 and 2; R, lanes 3 and 4) in combination with Myc-PEX19 or FLAG-PEX19 were lysed and subjected to immunoprecipitation with anti-FLAG agarose beads. Immunoprecipitates and input (10%) were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. Note that endogenous PEX19 is not visible at this exposure of the blot. (B and C) FLAG-tagged proteins indicated at the top were synthesized in RRL in the absence (B) or presence (C) of RRL-synthesized HA-PEX19 and then immunoprecipitated as in Fig. 5 A . Immunoprecipitates and input (10%) were analyzed by immunoblotting using the indicated antibodies. Solid and open arrowheads indicate unmodified and farnesylated HA-PEX19, respectively. Black line indicates that intervening lanes have been spliced out. IB, immunoblot; IP, immunoprecipitation.

    Techniques Used: Expressing, Immunoprecipitation, SDS Page, Synthesized

    TRC40 is not involved in the biogenesis of PEX26 in mammalian cells. (A) FLAG-tagged Sec61β and PEX26 were synthesized in RRL and subsequently subjected to immunoprecipitation (IP) with anti-FLAG agarose beads in detergent-free conditions. Immunoprecipitates and input (10%) were analyzed by SDS-PAGE and immunoblotting using the indicated antibodies. (B) pex19 ZP119 cells were transfected with ER-EGFP together with either FLAG-PEX26 (a–c) or FLAG-SEC61β (d and e). Cells were immunostained with antibodies against FLAG and cytochrome c (Cyt c). Bars, 10 µm.
    Figure Legend Snippet: TRC40 is not involved in the biogenesis of PEX26 in mammalian cells. (A) FLAG-tagged Sec61β and PEX26 were synthesized in RRL and subsequently subjected to immunoprecipitation (IP) with anti-FLAG agarose beads in detergent-free conditions. Immunoprecipitates and input (10%) were analyzed by SDS-PAGE and immunoblotting using the indicated antibodies. (B) pex19 ZP119 cells were transfected with ER-EGFP together with either FLAG-PEX26 (a–c) or FLAG-SEC61β (d and e). Cells were immunostained with antibodies against FLAG and cytochrome c (Cyt c). Bars, 10 µm.

    Techniques Used: Synthesized, Immunoprecipitation, SDS Page, Transfection

    PEX19 forms a soluble complex with PEX26 in the cytosol and maintains its import-competent state. (A, left) CHO-K1 cells transiently transfected with FLAG-PEX26 and either empty vector (lanes 1–3) or HA-PEX19 (lanes 4–6) were fractionated into postnuclear supernatant (T, total), cytosolic (S, supernatant), and organelle (P, pellet) fractions. Equal aliquots of the respective fractions were analyzed by SDS-PAGE and immunoblotting using the indicated antibodies. Lactate dehydrogenase (LDH) and PEX14, a PMP, are markers for supernatant and pellet fractions, respectively. (right) Supernatant fraction obtained from CHO-K1 cells coexpressing FLAG-PEX26 and HA-PEX19 was subjected to immunoprecipitation with the anti-HA antibody (lane 8) or preimmune serum (lane 9). Immunoprecipitates and input (10%; lane 7) were analyzed by immunoblotting using the indicated antibodies. (B) HeLa cells were semipermeabilized and incubated at 26°C for 1 h with the cytosolic fraction of CHO-K1 cells expressing either HA-PEX26 plus FLAG-PEX19 (a and b) or HA-PEX26 alone (c and d). Cells were immunostained with antibodies against HA and catalase, a peroxisomal matrix protein. (C) Cytosolic fractions of CHO-K1 cells coexpressing HA-PEX26 and either FLAG-PEX19 (lanes 1 and 3) or Myc-PEX19 (lanes 2 and 4) were subjected to immunoprecipitation with anti-FLAG agarose beads. Immunoprecipitates were eluted with FLAG peptides and analyzed by immunoblotting using the indicated antibodies. Input (10%) was loaded in lanes 1 and 2. (D) In vitro import assay was performed as in B using the eluted fraction shown in C (lane 3) containing FLAG-PEX19–HA-PEX26 complexes (a and b). Another eluted fraction shown in C (lane 4) was used as a control (c and d). Cells were immunostained as in B. (E) Cells shown in D (top) were treated with 0.1 M Na 2 CO 3 and separated into soluble (supernatant) and membrane (pellet) fractions. Equal aliquots of respective fractions were analyzed by immunoblotting with the indicated antibodies. PEX13, a PMP; acyl-CoA oxidase (AOx), a peroxisomal matrix enzyme. Of the three components of AOx (A, B, and C), only the B chain is shown. Solid and open arrowheads in A and C indicate unmodified and farnesylated epitope-tagged PEX19, respectively. IP, immunoprecipitation. Bars, 10 µm.
    Figure Legend Snippet: PEX19 forms a soluble complex with PEX26 in the cytosol and maintains its import-competent state. (A, left) CHO-K1 cells transiently transfected with FLAG-PEX26 and either empty vector (lanes 1–3) or HA-PEX19 (lanes 4–6) were fractionated into postnuclear supernatant (T, total), cytosolic (S, supernatant), and organelle (P, pellet) fractions. Equal aliquots of the respective fractions were analyzed by SDS-PAGE and immunoblotting using the indicated antibodies. Lactate dehydrogenase (LDH) and PEX14, a PMP, are markers for supernatant and pellet fractions, respectively. (right) Supernatant fraction obtained from CHO-K1 cells coexpressing FLAG-PEX26 and HA-PEX19 was subjected to immunoprecipitation with the anti-HA antibody (lane 8) or preimmune serum (lane 9). Immunoprecipitates and input (10%; lane 7) were analyzed by immunoblotting using the indicated antibodies. (B) HeLa cells were semipermeabilized and incubated at 26°C for 1 h with the cytosolic fraction of CHO-K1 cells expressing either HA-PEX26 plus FLAG-PEX19 (a and b) or HA-PEX26 alone (c and d). Cells were immunostained with antibodies against HA and catalase, a peroxisomal matrix protein. (C) Cytosolic fractions of CHO-K1 cells coexpressing HA-PEX26 and either FLAG-PEX19 (lanes 1 and 3) or Myc-PEX19 (lanes 2 and 4) were subjected to immunoprecipitation with anti-FLAG agarose beads. Immunoprecipitates were eluted with FLAG peptides and analyzed by immunoblotting using the indicated antibodies. Input (10%) was loaded in lanes 1 and 2. (D) In vitro import assay was performed as in B using the eluted fraction shown in C (lane 3) containing FLAG-PEX19–HA-PEX26 complexes (a and b). Another eluted fraction shown in C (lane 4) was used as a control (c and d). Cells were immunostained as in B. (E) Cells shown in D (top) were treated with 0.1 M Na 2 CO 3 and separated into soluble (supernatant) and membrane (pellet) fractions. Equal aliquots of respective fractions were analyzed by immunoblotting with the indicated antibodies. PEX13, a PMP; acyl-CoA oxidase (AOx), a peroxisomal matrix enzyme. Of the three components of AOx (A, B, and C), only the B chain is shown. Solid and open arrowheads in A and C indicate unmodified and farnesylated epitope-tagged PEX19, respectively. IP, immunoprecipitation. Bars, 10 µm.

    Techniques Used: Transfection, Plasmid Preparation, SDS Page, Immunoprecipitation, Incubation, Expressing, In Vitro

    Basic amino acid residues in the C segment are essential for peroxisomal targeting of PEX26. (A) Schematic representation of EGFP-PEX26 variants used. Amino acid sequences of the C segment are indicated by the single letter code. Basic and acidic amino acid residues are shown in red and blue letters, respectively, and mutated residues are underlined. (B) HeLa cells transiently expressing the respective EGFP-PEX26 proteins indicated on the left were fixed and then immunostained with the anti-PTS1 antibody. Bars, 10 µm. Intracellular localization of each EGFP-PEX26 protein is shown on the right. Per, peroxisome; Mito, mitochondria; marks (= and > ) represent equal frequency and higher frequency, respectively. For instance, the label “Mito Per > Per” for RRK3S indicates that cells showing both mitochondrial and peroxisomal localization of the EGFP-fused protein were found more frequently than cells showing only peroxisomal localization. (C) FLAG-PEX26 variants indicated at the top were synthesized in RRL in the presence of RRL-synthesized HA-PEX19 and subjected to immunoprecipitation with anti-FLAG agarose beads. Immunoprecipitates (IP) and input (10%) were analyzed by immunoblotting (IB) using the indicated antibodies. Solid and open arrowheads indicate unmodified and farnesylated HA-PEX19, respectively. The asterisk indicates a cross-reactive, nonspecific band.
    Figure Legend Snippet: Basic amino acid residues in the C segment are essential for peroxisomal targeting of PEX26. (A) Schematic representation of EGFP-PEX26 variants used. Amino acid sequences of the C segment are indicated by the single letter code. Basic and acidic amino acid residues are shown in red and blue letters, respectively, and mutated residues are underlined. (B) HeLa cells transiently expressing the respective EGFP-PEX26 proteins indicated on the left were fixed and then immunostained with the anti-PTS1 antibody. Bars, 10 µm. Intracellular localization of each EGFP-PEX26 protein is shown on the right. Per, peroxisome; Mito, mitochondria; marks (= and > ) represent equal frequency and higher frequency, respectively. For instance, the label “Mito Per > Per” for RRK3S indicates that cells showing both mitochondrial and peroxisomal localization of the EGFP-fused protein were found more frequently than cells showing only peroxisomal localization. (C) FLAG-PEX26 variants indicated at the top were synthesized in RRL in the presence of RRL-synthesized HA-PEX19 and subjected to immunoprecipitation with anti-FLAG agarose beads. Immunoprecipitates (IP) and input (10%) were analyzed by immunoblotting (IB) using the indicated antibodies. Solid and open arrowheads indicate unmodified and farnesylated HA-PEX19, respectively. The asterisk indicates a cross-reactive, nonspecific band.

    Techniques Used: Expressing, Synthesized, Immunoprecipitation

    25) Product Images from "Identification and molecular characterization of the G?12-Rho guanine nucleotide exchange factor pathway in Caenorhabditis elegans"

    Article Title: Identification and molecular characterization of the G?12-Rho guanine nucleotide exchange factor pathway in Caenorhabditis elegans

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

    doi: 10.1073/pnas.2533143100

    Biochemical interaction of the GPA-12–CeRhoGEF pathway. ( A ) CeRGS-RhoGEF interacts with activated GPA-12 in the presence of . Myc-tagged gpa-12 was cotransfected with the flag-tagged RGS domain of CeRhoGEF (CeRGS-RhoGEF) in COS-7 cells. GPA-12 was immunoprecipitated (IP) with anti-myc antibody from the cell lysates in the presence or absence of . The immunoprecipitates were separated on SDS/PAGE and immunoblotted with anti-flag ( Top ) or anti-myc (second panel from Top ) antibody. Expression of gpa-12 ( Bottom , with anti-myc antibody) or CeRGS-RhoGEF (second panel from Bottom , with anti-flag antibody) in lysates is shown. ( B ) Stimulation of SRF activity by the DH–PH domain of CeRhoGEF. COS-7 cells were transfected with SRE.L-luciferase reporter (0.5 μg), pCMV-β-galactosidase (0.5 μg), and the indicated expression plasmids, myc- LARG DH–PH (0.5 μg), myc- CeRhoGEF DH–PH (6 μg), or pEGFP-C3 (0.5 μg). SRF activities of cell lysates were measured as described in Methods . The expression of LARG DH–PH or CeRhoGEF DH–PH in lysates was detected by immunoblotting with anti-myc antibody ( Bottom ).
    Figure Legend Snippet: Biochemical interaction of the GPA-12–CeRhoGEF pathway. ( A ) CeRGS-RhoGEF interacts with activated GPA-12 in the presence of . Myc-tagged gpa-12 was cotransfected with the flag-tagged RGS domain of CeRhoGEF (CeRGS-RhoGEF) in COS-7 cells. GPA-12 was immunoprecipitated (IP) with anti-myc antibody from the cell lysates in the presence or absence of . The immunoprecipitates were separated on SDS/PAGE and immunoblotted with anti-flag ( Top ) or anti-myc (second panel from Top ) antibody. Expression of gpa-12 ( Bottom , with anti-myc antibody) or CeRGS-RhoGEF (second panel from Bottom , with anti-flag antibody) in lysates is shown. ( B ) Stimulation of SRF activity by the DH–PH domain of CeRhoGEF. COS-7 cells were transfected with SRE.L-luciferase reporter (0.5 μg), pCMV-β-galactosidase (0.5 μg), and the indicated expression plasmids, myc- LARG DH–PH (0.5 μg), myc- CeRhoGEF DH–PH (6 μg), or pEGFP-C3 (0.5 μg). SRF activities of cell lysates were measured as described in Methods . The expression of LARG DH–PH or CeRhoGEF DH–PH in lysates was detected by immunoblotting with anti-myc antibody ( Bottom ).

    Techniques Used: Immunoprecipitation, SDS Page, Expressing, Activity Assay, Transfection, Luciferase

    26) Product Images from "Integration of Calcium and Cyclic AMP Signaling Pathways by 14-3-3"

    Article Title: Integration of Calcium and Cyclic AMP Signaling Pathways by 14-3-3

    Journal: Molecular and Cellular Biology

    doi:

    Identification of the 14-3-3 binding site on NFAT. (A) Deletion analysis of NFAT3. The structure of NFAT3 is illustrated schematically. Flag-tagged NFAT3 proteins corresponding to residues 1 to 902, 1 to 580, 1 to 450, 1 to 365, 1 to 308, 1 to 260, 1 to 160, and 1 to 112 were expressed in COS cells and detected by immunoblot analysis of cell lysates with monoclonal antibody M2. The binding of NFAT3 and Raf-1 to immobilized GST–14-3-3τ was examined. Bound NFAT3 and Raf-1 were detected by immunoblot analysis. (B) Replacement of Ser-272, Ser-273, Ser-274, and Ser-289 with Ala decreases NFAT3 binding to 14-3-3. Epitope-tagged wild-type and mutated NFAT3 were expressed in COS cells and detected by immunoblot (IB) analysis of cell lysates with monoclonal antibody M2 (lower panel). The NFAT3 proteins were immunoprecipitated, and 14-3-3 present in the immunoprecipitates (IP) was detected by immunoblot analysis (upper panel; sizes are indicated in kilodaltons. (C) Immobilized GST–14-3-3τ was incubated with [ 35 S]methionine-labeled wild-type and mutated [Ala 272,273,274,289 ] NFAT3 prepared by in vitro translation. Control experiments were performed with in vitro-translated luciferase. Proteins in the lysate and bound to the immobilized GST–14-3-3τ were detected by autoradiography and quantitated by PhosphorImager analysis. (D) Epitope-tagged wild-type and mutated [Ala 272,273,274,289 ] NFAT3 were expressed in COS cells without (Control) and with an expression vector for the PKA catalytic subunit (PKA). NFAT3 proteins were immunoprecipitated, and 14-3-3 present in the immunoprecipitates (IP) was detected by immunoblot analysis (IB).
    Figure Legend Snippet: Identification of the 14-3-3 binding site on NFAT. (A) Deletion analysis of NFAT3. The structure of NFAT3 is illustrated schematically. Flag-tagged NFAT3 proteins corresponding to residues 1 to 902, 1 to 580, 1 to 450, 1 to 365, 1 to 308, 1 to 260, 1 to 160, and 1 to 112 were expressed in COS cells and detected by immunoblot analysis of cell lysates with monoclonal antibody M2. The binding of NFAT3 and Raf-1 to immobilized GST–14-3-3τ was examined. Bound NFAT3 and Raf-1 were detected by immunoblot analysis. (B) Replacement of Ser-272, Ser-273, Ser-274, and Ser-289 with Ala decreases NFAT3 binding to 14-3-3. Epitope-tagged wild-type and mutated NFAT3 were expressed in COS cells and detected by immunoblot (IB) analysis of cell lysates with monoclonal antibody M2 (lower panel). The NFAT3 proteins were immunoprecipitated, and 14-3-3 present in the immunoprecipitates (IP) was detected by immunoblot analysis (upper panel; sizes are indicated in kilodaltons. (C) Immobilized GST–14-3-3τ was incubated with [ 35 S]methionine-labeled wild-type and mutated [Ala 272,273,274,289 ] NFAT3 prepared by in vitro translation. Control experiments were performed with in vitro-translated luciferase. Proteins in the lysate and bound to the immobilized GST–14-3-3τ were detected by autoradiography and quantitated by PhosphorImager analysis. (D) Epitope-tagged wild-type and mutated [Ala 272,273,274,289 ] NFAT3 were expressed in COS cells without (Control) and with an expression vector for the PKA catalytic subunit (PKA). NFAT3 proteins were immunoprecipitated, and 14-3-3 present in the immunoprecipitates (IP) was detected by immunoblot analysis (IB).

    Techniques Used: Binding Assay, Immunoprecipitation, Incubation, Labeling, In Vitro, Luciferase, Autoradiography, Expressing, Plasmid Preparation

    NFAT interacts with 14-3-3. (A) Immobilized GST and GST-NFAT3 (residues 1 to 308) were incubated with extracts prepared from [ 35 S]methionine-labeled BHK cells. Bound proteins were separated by SDS-PAGE and detected by autoradiography. (B) Recombinant GST and GST-NFAT3 (residues 1 to 308) were phosphorylated by incubation with 1 mM ATP and purified PKA catalytic subunit. Immobilized GST and GST-NFAT3 were incubated with extracts prepared from Jurkat T cells. Bound 14-3-3 was detected by immunoblot (IB) analysis. The GST and GST-NFAT3 fusion proteins were detected by staining with Coomassie blue. (C) NFATp interacts with 14-3-3 in vivo. NFATp was immunoprecipitated from Jurkat T cell extracts by using a rabbit antibody to NFATp. Preimmune antibody was used as a control. 14-3-3 in the immunoprecipitates (IP) was detected by immunoblot (IB) analysis. (D) NFAT proteins bind 14-3-3. NFAT3, NFAT4, NFATp and NFATc were expressed in COS cells. NFATp was immunoprecipitated with an antibody to NFATp. Epitope-tagged NFAT3, NFAT4, and NFATc were immunoprecipitated with the anti-Flag monoclonal antibody (Ab) M2. 14-3-3 in the cells lysate and in the immunoprecipitates (IP) was detected by immunoblot (IB) analysis. Extracts prepared from mock-transfected cells were used as a control. IgG, immunoglobulin G.
    Figure Legend Snippet: NFAT interacts with 14-3-3. (A) Immobilized GST and GST-NFAT3 (residues 1 to 308) were incubated with extracts prepared from [ 35 S]methionine-labeled BHK cells. Bound proteins were separated by SDS-PAGE and detected by autoradiography. (B) Recombinant GST and GST-NFAT3 (residues 1 to 308) were phosphorylated by incubation with 1 mM ATP and purified PKA catalytic subunit. Immobilized GST and GST-NFAT3 were incubated with extracts prepared from Jurkat T cells. Bound 14-3-3 was detected by immunoblot (IB) analysis. The GST and GST-NFAT3 fusion proteins were detected by staining with Coomassie blue. (C) NFATp interacts with 14-3-3 in vivo. NFATp was immunoprecipitated from Jurkat T cell extracts by using a rabbit antibody to NFATp. Preimmune antibody was used as a control. 14-3-3 in the immunoprecipitates (IP) was detected by immunoblot (IB) analysis. (D) NFAT proteins bind 14-3-3. NFAT3, NFAT4, NFATp and NFATc were expressed in COS cells. NFATp was immunoprecipitated with an antibody to NFATp. Epitope-tagged NFAT3, NFAT4, and NFATc were immunoprecipitated with the anti-Flag monoclonal antibody (Ab) M2. 14-3-3 in the cells lysate and in the immunoprecipitates (IP) was detected by immunoblot (IB) analysis. Extracts prepared from mock-transfected cells were used as a control. IgG, immunoglobulin G.

    Techniques Used: Incubation, Labeling, SDS Page, Autoradiography, Recombinant, Purification, Staining, In Vivo, Immunoprecipitation, Transfection

    Calcium and cAMP regulate NFAT binding to 14-3-3. (A) Jurkat T cells were incubated without and with dibutyryl cAMP (500 μM) plus IBMX (50 μM) for 20 min prior to treatment with ionomycin (2 μM, 20 min) or with ionomycin plus PMA (100 nM, 20 min). NFATp proteins were immunoprecipitated, and 14-3-3 present in the immunoprecipitates (IP) was detected by immunoblot analysis (IB). (B) Jurkat T cells were incubated without and with CsA (100 ng/ml, 20 min) prior to treatment with ionomycin (2 μM, 20 min). Extracts prepared from the Jurkat T cells were incubated with immobilized GST–14-3-3τ. Bound NFATp and Raf-1 were detected by immunoblot analysis. The presence of equal amounts of NFATp in the lysates was confirmed by immunoblot analysis. The amount of GST–14-3-3τ was confirmed by Coomassie blue staining. (C) Jurkat T cells were incubated without and with dibutyryl cAMP (500 μM) plus IBMX (50 μM) for 20 min prior to treatment with ionomycin (2 μM, 20 min). Extracts prepared from the Jurkat T cells were incubated with immobilized GST–14-3-3τ. Bound NFATp and Raf-1 were detected by immunoblot analysis. The presence of equal amounts of NFATp in the lysates was confirmed by immunoblot analysis.
    Figure Legend Snippet: Calcium and cAMP regulate NFAT binding to 14-3-3. (A) Jurkat T cells were incubated without and with dibutyryl cAMP (500 μM) plus IBMX (50 μM) for 20 min prior to treatment with ionomycin (2 μM, 20 min) or with ionomycin plus PMA (100 nM, 20 min). NFATp proteins were immunoprecipitated, and 14-3-3 present in the immunoprecipitates (IP) was detected by immunoblot analysis (IB). (B) Jurkat T cells were incubated without and with CsA (100 ng/ml, 20 min) prior to treatment with ionomycin (2 μM, 20 min). Extracts prepared from the Jurkat T cells were incubated with immobilized GST–14-3-3τ. Bound NFATp and Raf-1 were detected by immunoblot analysis. The presence of equal amounts of NFATp in the lysates was confirmed by immunoblot analysis. The amount of GST–14-3-3τ was confirmed by Coomassie blue staining. (C) Jurkat T cells were incubated without and with dibutyryl cAMP (500 μM) plus IBMX (50 μM) for 20 min prior to treatment with ionomycin (2 μM, 20 min). Extracts prepared from the Jurkat T cells were incubated with immobilized GST–14-3-3τ. Bound NFATp and Raf-1 were detected by immunoblot analysis. The presence of equal amounts of NFATp in the lysates was confirmed by immunoblot analysis.

    Techniques Used: Binding Assay, Incubation, Immunoprecipitation, Staining

    27) Product Images from "N-Glycans of F Protein Differentially Affect Fusion Activity of Human Respiratory Syncytial Virus"

    Article Title: N-Glycans of F Protein Differentially Affect Fusion Activity of Human Respiratory Syncytial Virus

    Journal: Journal of Virology

    doi: 10.1128/JVI.75.10.4744-4751.2001

    Electrophoretic mobilities of the F protein mutants. MVA-T7-infected BSR-T7/5 cells were transfected with recombinant pTM1 plasmids (lane a, parental F; lane b, N27Q; lane c, N70Q; lane d, N27/70Q; lane e, N116Q; lane f, N120Q; lane g, N116/120Q; lane h, N126Q; lane i, N500Q; lane j, N27/500Q; lane k, N70/500Q; lane l, N27/70/500Q; and lane m, pTM1). The cells were metabolically labeled with [ 35 S]methionine-[ 35 S]cysteine, F protein was immunoprecipitated from the cell lysates, and the immunoprecipitates were separated by Tricine–SDS–10% polyacrylamide gel electrophoresis under reducing conditions. The relative positions of standard proteins of the indicated molecular masses (in kilodaltons) are shown on the left.
    Figure Legend Snippet: Electrophoretic mobilities of the F protein mutants. MVA-T7-infected BSR-T7/5 cells were transfected with recombinant pTM1 plasmids (lane a, parental F; lane b, N27Q; lane c, N70Q; lane d, N27/70Q; lane e, N116Q; lane f, N120Q; lane g, N116/120Q; lane h, N126Q; lane i, N500Q; lane j, N27/500Q; lane k, N70/500Q; lane l, N27/70/500Q; and lane m, pTM1). The cells were metabolically labeled with [ 35 S]methionine-[ 35 S]cysteine, F protein was immunoprecipitated from the cell lysates, and the immunoprecipitates were separated by Tricine–SDS–10% polyacrylamide gel electrophoresis under reducing conditions. The relative positions of standard proteins of the indicated molecular masses (in kilodaltons) are shown on the left.

    Techniques Used: Infection, Transfection, Recombinant, Metabolic Labelling, Labeling, Immunoprecipitation, Polyacrylamide Gel Electrophoresis

    Detection of cell surface-biotinylated F protein. Transfected BSR-T7/5 cells were labeled with sulfo-NHS-biotin at 4°C, and F protein was immunoprecipitated from the cell lysates by using a monoclonal antibody. The immunoprecipitates were separated by SDS-polyacrylamide gel electrophoresis under reducing conditions, transferred to nitrocellulose membranes, and probed with streptavidin-peroxidase. (A) Lanes a and m, parental F; lane b, N27/70/500Q; lane c, N27/500Q; lane d, N70/500Q; lane e, 27/70Q; lane f, N500Q; lane g, N27Q; lane h, N70Q; lane i, N116Q; lane j, N120Q; lane k, N116/120Q; lane l, N126Q). (B) Lane a, N500A; lane b, S502A; lane c, N500Q; lane d, parental F. The relative positions of standard proteins of the indicated molecular masses (in kilodaltons) are shown on the left.
    Figure Legend Snippet: Detection of cell surface-biotinylated F protein. Transfected BSR-T7/5 cells were labeled with sulfo-NHS-biotin at 4°C, and F protein was immunoprecipitated from the cell lysates by using a monoclonal antibody. The immunoprecipitates were separated by SDS-polyacrylamide gel electrophoresis under reducing conditions, transferred to nitrocellulose membranes, and probed with streptavidin-peroxidase. (A) Lanes a and m, parental F; lane b, N27/70/500Q; lane c, N27/500Q; lane d, N70/500Q; lane e, 27/70Q; lane f, N500Q; lane g, N27Q; lane h, N70Q; lane i, N116Q; lane j, N120Q; lane k, N116/120Q; lane l, N126Q). (B) Lane a, N500A; lane b, S502A; lane c, N500Q; lane d, parental F. The relative positions of standard proteins of the indicated molecular masses (in kilodaltons) are shown on the left.

    Techniques Used: Transfection, Labeling, Immunoprecipitation, Polyacrylamide Gel Electrophoresis

    28) Product Images from "Rab11 mediates selective recycling and endocytic trafficking in Trypanosoma brucei"

    Article Title: Rab11 mediates selective recycling and endocytic trafficking in Trypanosoma brucei

    Journal: Traffic (Copenhagen, Denmark)

    doi: 10.1111/tra.12565

    Rab11 depletion does not affect the turnover of transferrin receptor A. TbRab11 silencing was induced for the indicated times and TfR levels were assessed by immunoblotting with anti-TfR (10 7 cell equivalents per lane), and with anti-BiP as a loading control. A representative LICOR image is presented. Mobilities of ESAG6 (E6), ESAG7 (E7) subunits, and BiP are indicated on the left; mobilities of molecular mass markers are indicated on the right (kDa). B. Control and TbRab11 silenced cells were used subjected to pulse/chase (15 min/4 hr) metabolic radiolabeling with [ 35 S]Met/Cys. As indicated FMK024 (20 μM) was included to block lysosomal thiol proteases. At the indicted times cell extracts were prepared and functional transferrin receptor was pulled down using transferrin beads. Immunoprecipitates were analyzed by SDS-PAGE/phosphorimaging. Top. Representative phosphorimages for control (Tet−) and silenced (Tet+) cells are presented (10 7 cell equivalents per lane). ESAG7, E7; immature ESAG6 (E6 i ); mature ESAG6 (E6 m ). Bottom. ESAG7 signals were quantified and normalized to values at 0.5 hours (mean ± std. dev., n=4 biological replicates). C. TfR localization was determined in fixed permeabilized control and silenced cells by immunofluorescence with anti-TfR antibody (green). The lysosome (l) was visualized with mAb anti-p67 (red); nuclei and kinetoplasts (n, k) with DAPI. Representative deconvolved summed stack images are presented. The position of the flagellar pocket is indicated by opposing arrowheads. Scale bar: 2 μm.
    Figure Legend Snippet: Rab11 depletion does not affect the turnover of transferrin receptor A. TbRab11 silencing was induced for the indicated times and TfR levels were assessed by immunoblotting with anti-TfR (10 7 cell equivalents per lane), and with anti-BiP as a loading control. A representative LICOR image is presented. Mobilities of ESAG6 (E6), ESAG7 (E7) subunits, and BiP are indicated on the left; mobilities of molecular mass markers are indicated on the right (kDa). B. Control and TbRab11 silenced cells were used subjected to pulse/chase (15 min/4 hr) metabolic radiolabeling with [ 35 S]Met/Cys. As indicated FMK024 (20 μM) was included to block lysosomal thiol proteases. At the indicted times cell extracts were prepared and functional transferrin receptor was pulled down using transferrin beads. Immunoprecipitates were analyzed by SDS-PAGE/phosphorimaging. Top. Representative phosphorimages for control (Tet−) and silenced (Tet+) cells are presented (10 7 cell equivalents per lane). ESAG7, E7; immature ESAG6 (E6 i ); mature ESAG6 (E6 m ). Bottom. ESAG7 signals were quantified and normalized to values at 0.5 hours (mean ± std. dev., n=4 biological replicates). C. TfR localization was determined in fixed permeabilized control and silenced cells by immunofluorescence with anti-TfR antibody (green). The lysosome (l) was visualized with mAb anti-p67 (red); nuclei and kinetoplasts (n, k) with DAPI. Representative deconvolved summed stack images are presented. The position of the flagellar pocket is indicated by opposing arrowheads. Scale bar: 2 μm.

    Techniques Used: Pulse Chase, Radioactivity, Blocking Assay, Functional Assay, SDS Page, Immunofluorescence

    29) Product Images from "The linkage between ?1 integrin and the actin cytoskeleton is differentially regulated by tyrosine and serine/threonine phosphorylation of ?1 integrin in normal and cancerous human breast cells"

    Article Title: The linkage between ?1 integrin and the actin cytoskeleton is differentially regulated by tyrosine and serine/threonine phosphorylation of ?1 integrin in normal and cancerous human breast cells

    Journal: BMC Cell Biology

    doi: 10.1186/1471-2121-2-23

    Effects of dephosphorylation of β1 integrin on its coprecipitation with actin. (A) The β1 integrin immunoprecipitates from quiescent HBE or MCF-7 cells were treated with (+) or without (-) PTP, then immunoblotted with anti-actin, anti-β1 integrin, or anti-PY antibody. The PTP-treated β1 integrin immunoprecipitates from MCF-7 cells were incubated for 30 min with (+) or without (-) the supernatant of the β1 immunoprecipitates containing unbound actin (Cell lysate), or with 5 μg/ml exogenous human platelet actin (A). Molecular size markers are indicated at right in kDa. (B) The β1 integrin immunoprecipitates from quiescent HBE or MCF-7 cells were treated with (+) or without (-) PP2A 1 , then the PP2A 1 -treated β1 integrin from MCF-7 cells were incubated for 30 min with (+) or without (-) the supernatant of the β1 immunoprecipitates (Cell lysate), or with 5 μg/ml exogenous human platelet actin (A). The immunoblots were probed with anti-actin, anti-β1 integrin, or anti-PY antibody. Molecular size markers are indicated at right in kDa.
    Figure Legend Snippet: Effects of dephosphorylation of β1 integrin on its coprecipitation with actin. (A) The β1 integrin immunoprecipitates from quiescent HBE or MCF-7 cells were treated with (+) or without (-) PTP, then immunoblotted with anti-actin, anti-β1 integrin, or anti-PY antibody. The PTP-treated β1 integrin immunoprecipitates from MCF-7 cells were incubated for 30 min with (+) or without (-) the supernatant of the β1 immunoprecipitates containing unbound actin (Cell lysate), or with 5 μg/ml exogenous human platelet actin (A). Molecular size markers are indicated at right in kDa. (B) The β1 integrin immunoprecipitates from quiescent HBE or MCF-7 cells were treated with (+) or without (-) PP2A 1 , then the PP2A 1 -treated β1 integrin from MCF-7 cells were incubated for 30 min with (+) or without (-) the supernatant of the β1 immunoprecipitates (Cell lysate), or with 5 μg/ml exogenous human platelet actin (A). The immunoblots were probed with anti-actin, anti-β1 integrin, or anti-PY antibody. Molecular size markers are indicated at right in kDa.

    Techniques Used: De-Phosphorylation Assay, Incubation, Western Blot

    Coprecipitation of α-actinin, but not talin or vinculin, with β1 integrin in HBE and MCF-7 cells. (A) Integrin β1 was immunoprecipitated (IP) from HBE and MCF-7 cells using the anti-β1 integrin antibody or control IgG. The immunoprecipitates were probed with anti-α-actinin, anti-talin, or anti-vinculin antibody. Molecular size markers are indicated at right in kDa. (B) Equal amounts (5 μg protein) of whole cell lysates from quiescent HBE and MCF-7 cells were probed with anti-α-actinin, anti-talin, or anti-vinculin antibody. Molecular size markers are indicated at right in kDa.
    Figure Legend Snippet: Coprecipitation of α-actinin, but not talin or vinculin, with β1 integrin in HBE and MCF-7 cells. (A) Integrin β1 was immunoprecipitated (IP) from HBE and MCF-7 cells using the anti-β1 integrin antibody or control IgG. The immunoprecipitates were probed with anti-α-actinin, anti-talin, or anti-vinculin antibody. Molecular size markers are indicated at right in kDa. (B) Equal amounts (5 μg protein) of whole cell lysates from quiescent HBE and MCF-7 cells were probed with anti-α-actinin, anti-talin, or anti-vinculin antibody. Molecular size markers are indicated at right in kDa.

    Techniques Used: Immunoprecipitation

    The phosphorylation state of β1 integrin. (A) HBE or MCF-7 cells which were quiescent and adherent to collagen IV were metabolically labeled with [ 32 P]orthophosphoric acid and β1 integrin was immunoprecipitated from the cells. After separation on 8% SDS-PAGE, gels were dried and autoradiographed. Unlabeled samples were electrophoresed and transferred onto membranes. The blots were probed with antibodies to β1 integrin and α-actinin. Molecular size markers are indicated at right in kDa. (B) Tyrosine phosphorylation of β1 integrin in HBE and MCF-7 cells. Integrin β1 was immunoprecipitated from HBE or MCF-7 cells and the immunoprecipitates were resolved by 8% SDS-PAGE before blotting. The blots were then probed with anti-PY or anti-integrin β1 monoclonal antibody. Molecular size markers are indicated at right in kDa.
    Figure Legend Snippet: The phosphorylation state of β1 integrin. (A) HBE or MCF-7 cells which were quiescent and adherent to collagen IV were metabolically labeled with [ 32 P]orthophosphoric acid and β1 integrin was immunoprecipitated from the cells. After separation on 8% SDS-PAGE, gels were dried and autoradiographed. Unlabeled samples were electrophoresed and transferred onto membranes. The blots were probed with antibodies to β1 integrin and α-actinin. Molecular size markers are indicated at right in kDa. (B) Tyrosine phosphorylation of β1 integrin in HBE and MCF-7 cells. Integrin β1 was immunoprecipitated from HBE or MCF-7 cells and the immunoprecipitates were resolved by 8% SDS-PAGE before blotting. The blots were then probed with anti-PY or anti-integrin β1 monoclonal antibody. Molecular size markers are indicated at right in kDa.

    Techniques Used: Metabolic Labelling, Labeling, Immunoprecipitation, SDS Page

    CaMKII binding to β1 integrin in MCF-7 cells. (A) The β1 integrin immunoprecipitates (IP) with the anti-β1 integrin antibody or control IgG from HBE or MCF-7 cells were probed with antibody to all CaMKII isoforms or anti-β1 integrin antibody. Molecular size markers are indicated at right in kDa. (B) The β1 integrin immunoprecipitates from HBE or MCF-7 cells were incubated with the substrate peptide Autocamtide II, calmodulin, inhibitor peptides of PKA and PKC, and [ 32 P]ATP with (closed columns) or without (open columns) 10 μM KN-62 at 30°C for 10 min. After subtraction of the background, the activity was normalized to the amount of β1 integrin and expressed relative to that of the β1 integrin immunoprecipitates from HBE cells without KN-62. Results represent the mean ± S.D. (bars) of duplicate assays of three independent experiments.
    Figure Legend Snippet: CaMKII binding to β1 integrin in MCF-7 cells. (A) The β1 integrin immunoprecipitates (IP) with the anti-β1 integrin antibody or control IgG from HBE or MCF-7 cells were probed with antibody to all CaMKII isoforms or anti-β1 integrin antibody. Molecular size markers are indicated at right in kDa. (B) The β1 integrin immunoprecipitates from HBE or MCF-7 cells were incubated with the substrate peptide Autocamtide II, calmodulin, inhibitor peptides of PKA and PKC, and [ 32 P]ATP with (closed columns) or without (open columns) 10 μM KN-62 at 30°C for 10 min. After subtraction of the background, the activity was normalized to the amount of β1 integrin and expressed relative to that of the β1 integrin immunoprecipitates from HBE cells without KN-62. Results represent the mean ± S.D. (bars) of duplicate assays of three independent experiments.

    Techniques Used: Binding Assay, Incubation, Activity Assay

    Coprecipitation of actin with β1 integrin in HBE cells and not in MCF-7 cells. (A) Integrin β1 was immunoprecipitated (IP) with the anti-β1 integrin antibody or control IgG from HBE or MCF-7 cells which were quiescent and adherent to collagen IV. The immunoprecipitates were immunoblotted with anti-actin or anti-integrin β1 monoclonal antibody. Molecular size markers are indicated at right in kDa. (B) Whole cell lysates were prepared from HBE or MCF-7 cells which were quiescent and adherent to collagen IV, and equal amounts (5 μ g protein) of the lysates were resolved by SDS-PAGE and blotted. The blots were probed with anti-actin or anti-integrin β1 monoclonal antibody. Molecular size markers are indicated at right in kDa.
    Figure Legend Snippet: Coprecipitation of actin with β1 integrin in HBE cells and not in MCF-7 cells. (A) Integrin β1 was immunoprecipitated (IP) with the anti-β1 integrin antibody or control IgG from HBE or MCF-7 cells which were quiescent and adherent to collagen IV. The immunoprecipitates were immunoblotted with anti-actin or anti-integrin β1 monoclonal antibody. Molecular size markers are indicated at right in kDa. (B) Whole cell lysates were prepared from HBE or MCF-7 cells which were quiescent and adherent to collagen IV, and equal amounts (5 μ g protein) of the lysates were resolved by SDS-PAGE and blotted. The blots were probed with anti-actin or anti-integrin β1 monoclonal antibody. Molecular size markers are indicated at right in kDa.

    Techniques Used: Immunoprecipitation, SDS Page

    30) Product Images from "CDK phosphorylation of Xenopus laevis M18BP1 promotes its metaphase centromere localization"

    Article Title: CDK phosphorylation of Xenopus laevis M18BP1 promotes its metaphase centromere localization

    Journal: bioRxiv

    doi: 10.1101/355487

    Identification of phosphosites in M18BP1-1 161-580 by mass spectrometry A) Pull-downs from metaphase or interphase extract depleted of M18BP1 with (+) or without (−) MBP-M18BP1-1 161-580 . (Top) Immunoblot showing that MBP-M18BP1-1 161-580 specifically co-immunoprecipitates CENP-C from metaphase extract. (Bottom) Coomassie colloidal blue-stained gel showing material precipitated with α-MBP antibody-coated beads. Red boxes indicate bands that were excised and submitted for mass spectrometry. B) Immunoblot showing levels of MBP-M18BP1-1 161-580 in M18BP1-depleted extract relative to endogenous M18BP1 levels (Undepleted, left lane). Nonspecific band recognized by α-M18BP1 antibody indicated by asterisk. MBP-M18BP1-1 161-580 was ~56-fold in excess of endogenous M18BP1.
    Figure Legend Snippet: Identification of phosphosites in M18BP1-1 161-580 by mass spectrometry A) Pull-downs from metaphase or interphase extract depleted of M18BP1 with (+) or without (−) MBP-M18BP1-1 161-580 . (Top) Immunoblot showing that MBP-M18BP1-1 161-580 specifically co-immunoprecipitates CENP-C from metaphase extract. (Bottom) Coomassie colloidal blue-stained gel showing material precipitated with α-MBP antibody-coated beads. Red boxes indicate bands that were excised and submitted for mass spectrometry. B) Immunoblot showing levels of MBP-M18BP1-1 161-580 in M18BP1-depleted extract relative to endogenous M18BP1 levels (Undepleted, left lane). Nonspecific band recognized by α-M18BP1 antibody indicated by asterisk. MBP-M18BP1-1 161-580 was ~56-fold in excess of endogenous M18BP1.

    Techniques Used: Mass Spectrometry, Staining

    31) Product Images from "Rsu1 contributes to regulation of cell adhesion and spreading by PINCH1-dependent and - independent mechanisms"

    Article Title: Rsu1 contributes to regulation of cell adhesion and spreading by PINCH1-dependent and - independent mechanisms

    Journal: Journal of Cell Communication and Signaling

    doi: 10.1007/s12079-013-0207-5

    Rsu1 - PINCH1 interaction is required to promote the formation of mature focal adhesions in MCF10A cells. MCF10A cell lines infected with pBABE control vector or expressing wt-Rsu1-myc or N92D-Rsu1-myc were depleted of endogenous Rsu1 by transfection with endogenous Rsu1 specific siRNA. The cells were plated on fibronectin coverslips and compared to those transfected with negative control siRNA. a . Cells were fixed at 96 h post transfection and assayed for Rsu1 by immunofluorescence b . Cells were fixed and stained with anti-myc tag and anti-vinculin antibodies. Nuclei were counterstained with DAPI. Scale bar 10 μm. Zen 2009 software was used for fluorescence intensity measurements. Fluorescence values are expressed in arbitrary units. c . Lysates of 293 T cells cotransfected with plasmid encoding HA-PINCH1 and wt-Rsu1-myc or N92D-Rsu1-myc were immunoprecipitaed with anti-HA (rat) antibody. The immunoprecipitates and sample of lysates were analyzed by western blot with anti-myc tag (mouse) antibody for presence of myc-tagged Rsu1. The blot was subsequently reacted with antibody to Rsu1 to demonstrate that the endogenous Rsu1 co-precipitated with HA-PINCH1. d . Lysates from MCF10A cells infected with pBABE, wt-Rsu1-myc or N92D-Rsu1-myc were immunoprecipitated with anti-myc IgG (rabbit) cross linked to agarose beads. The immunoprecipitates and sample of lysates were analyzed by western blot with anti-PINCH (mouse) antibody for presence of co-precipitated endogenous PINCH1 and anti-Rsu1 (rabbit) for endogenous and myc-tagged Rsu1
    Figure Legend Snippet: Rsu1 - PINCH1 interaction is required to promote the formation of mature focal adhesions in MCF10A cells. MCF10A cell lines infected with pBABE control vector or expressing wt-Rsu1-myc or N92D-Rsu1-myc were depleted of endogenous Rsu1 by transfection with endogenous Rsu1 specific siRNA. The cells were plated on fibronectin coverslips and compared to those transfected with negative control siRNA. a . Cells were fixed at 96 h post transfection and assayed for Rsu1 by immunofluorescence b . Cells were fixed and stained with anti-myc tag and anti-vinculin antibodies. Nuclei were counterstained with DAPI. Scale bar 10 μm. Zen 2009 software was used for fluorescence intensity measurements. Fluorescence values are expressed in arbitrary units. c . Lysates of 293 T cells cotransfected with plasmid encoding HA-PINCH1 and wt-Rsu1-myc or N92D-Rsu1-myc were immunoprecipitaed with anti-HA (rat) antibody. The immunoprecipitates and sample of lysates were analyzed by western blot with anti-myc tag (mouse) antibody for presence of myc-tagged Rsu1. The blot was subsequently reacted with antibody to Rsu1 to demonstrate that the endogenous Rsu1 co-precipitated with HA-PINCH1. d . Lysates from MCF10A cells infected with pBABE, wt-Rsu1-myc or N92D-Rsu1-myc were immunoprecipitated with anti-myc IgG (rabbit) cross linked to agarose beads. The immunoprecipitates and sample of lysates were analyzed by western blot with anti-PINCH (mouse) antibody for presence of co-precipitated endogenous PINCH1 and anti-Rsu1 (rabbit) for endogenous and myc-tagged Rsu1

    Techniques Used: Infection, Plasmid Preparation, Expressing, Transfection, Negative Control, Immunofluorescence, Staining, Software, Fluorescence, Western Blot, Immunoprecipitation

    32) Product Images from "CD72 negatively regulates B lymphocyte responses to the lupus-related endogenous toll-like receptor 7 ligand Sm/RNP"

    Article Title: CD72 negatively regulates B lymphocyte responses to the lupus-related endogenous toll-like receptor 7 ligand Sm/RNP

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20160560

    NP-Sm/RNP induces phosphorylation and SHP-1 recruitment of CD72 in NP-reactive B cells. (A–D) The B cell line BAL17-9T13 that expresses anti-NP BCR was treated with NP-BSA, NP-Sm/RNP, or a combination of NP-BSA and Sm/RNP for the indicated time, and lysates were immunoprecipitated (IP) with anti-CD72 antibody. Phosphorylated CD72 (pCD72; A) and SHP-1 (C) in the immunoprecipitates were analyzed by Western blotting. The intensities of protein bands for total CD72 (B and D), phosphorylated CD72 (B), and SHP-1 (D) were quantified and expressed as fold-change relative to unstimulated cells (0 min). Data are representative of four independent experiments. (E) Schematic representation of the mechanisms for CD72-mediated signal regulation. Coligation of CD72 with BCR mediated by Sm/RNP (left) but not independent ligation of CD72 and BCR (right) induces CD72 phosphorylation by BCR-associated kinases such as Lyn. The requirement of coligation restricts CD72-mediated signal inhibition only in Sm/RNP-reactive B cells. P, phosphate group; Y, tyrosine residue.
    Figure Legend Snippet: NP-Sm/RNP induces phosphorylation and SHP-1 recruitment of CD72 in NP-reactive B cells. (A–D) The B cell line BAL17-9T13 that expresses anti-NP BCR was treated with NP-BSA, NP-Sm/RNP, or a combination of NP-BSA and Sm/RNP for the indicated time, and lysates were immunoprecipitated (IP) with anti-CD72 antibody. Phosphorylated CD72 (pCD72; A) and SHP-1 (C) in the immunoprecipitates were analyzed by Western blotting. The intensities of protein bands for total CD72 (B and D), phosphorylated CD72 (B), and SHP-1 (D) were quantified and expressed as fold-change relative to unstimulated cells (0 min). Data are representative of four independent experiments. (E) Schematic representation of the mechanisms for CD72-mediated signal regulation. Coligation of CD72 with BCR mediated by Sm/RNP (left) but not independent ligation of CD72 and BCR (right) induces CD72 phosphorylation by BCR-associated kinases such as Lyn. The requirement of coligation restricts CD72-mediated signal inhibition only in Sm/RNP-reactive B cells. P, phosphate group; Y, tyrosine residue.

    Techniques Used: Immunoprecipitation, Western Blot, Ligation, Inhibition

    33) Product Images from "The interactome of a PTB domain-containing adapter protein, Odin, revealed by SILAC"

    Article Title: The interactome of a PTB domain-containing adapter protein, Odin, revealed by SILAC

    Journal: Journal of proteomics

    doi: 10.1016/j.jprot.2010.11.006

    Validation of protein-protein interactions by co-immunoprecipitation experiments. The Odin protein complex was harvested by immunoprecipitation with anti-FLAG antibodies. The cell lysates and corresponding immunoprecipitates were resolved by SDS-PAGE
    Figure Legend Snippet: Validation of protein-protein interactions by co-immunoprecipitation experiments. The Odin protein complex was harvested by immunoprecipitation with anti-FLAG antibodies. The cell lysates and corresponding immunoprecipitates were resolved by SDS-PAGE

    Techniques Used: Immunoprecipitation, SDS Page

    34) Product Images from "PGAP2 Is Essential for Correct Processing and Stable Expression of GPI-anchored Proteins D⃞"

    Article Title: PGAP2 Is Essential for Correct Processing and Stable Expression of GPI-anchored Proteins D⃞

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E05-11-1005

    Intracellular processing of a reporter GPI-AP. (A) Wild-type 3B2A (lanes 1–3) and C84 (lanes 4 and 5) cells were transfected without (lane 1) or with (lanes 2–5) VSVGts-FF-mEGFP-GPI and cultured at 40°C for 1 d. Cells were further cultured under the conditions shown on the right. The cell lysates were immunoprecipitated with an anti-FLAG antibody, subjected to SDS-PAGE, and analyzed by Western blotting. a, VSVGts-FF-mEGFP-GPI; b, FLAG-mEGFP-GPI (a cleavage product of VSVGts-FF-mEGFP-GPI by furin); asterisk, a degradation product. (B) 3B2A and C84 cells transfected with VSVGts-FF-mEGFP-GPI were cultured under the same conditions shown in lanes 3 and 5 in A. FLAG-mEGFP-GPI was collected from the cell lysates with anti-FLAG beads and eluted with a FLAG-peptide. To prepare lyso-GPI-anchored FLAG-mEGFP-GPI, aliquots of the immunoprecipitates from 3B2A cells were treated with PLA 2 . FLAG-mEGFP-GPIs were chromatographed in an Octyl-FF column with a 5–40% gradient of 1-propanol. Fractions were subjected to SDS-PAGE and analyzed by Western blotting with an anti-FLAG antibody.
    Figure Legend Snippet: Intracellular processing of a reporter GPI-AP. (A) Wild-type 3B2A (lanes 1–3) and C84 (lanes 4 and 5) cells were transfected without (lane 1) or with (lanes 2–5) VSVGts-FF-mEGFP-GPI and cultured at 40°C for 1 d. Cells were further cultured under the conditions shown on the right. The cell lysates were immunoprecipitated with an anti-FLAG antibody, subjected to SDS-PAGE, and analyzed by Western blotting. a, VSVGts-FF-mEGFP-GPI; b, FLAG-mEGFP-GPI (a cleavage product of VSVGts-FF-mEGFP-GPI by furin); asterisk, a degradation product. (B) 3B2A and C84 cells transfected with VSVGts-FF-mEGFP-GPI were cultured under the same conditions shown in lanes 3 and 5 in A. FLAG-mEGFP-GPI was collected from the cell lysates with anti-FLAG beads and eluted with a FLAG-peptide. To prepare lyso-GPI-anchored FLAG-mEGFP-GPI, aliquots of the immunoprecipitates from 3B2A cells were treated with PLA 2 . FLAG-mEGFP-GPIs were chromatographed in an Octyl-FF column with a 5–40% gradient of 1-propanol. Fractions were subjected to SDS-PAGE and analyzed by Western blotting with an anti-FLAG antibody.

    Techniques Used: Transfection, Cell Culture, Immunoprecipitation, SDS Page, Western Blot, Proximity Ligation Assay

    35) Product Images from "The Tumor Suppressor CYLD Interacts with TRIP and Regulates Negatively Nuclear Factor ?B Activation by Tumor Necrosis Factor"

    Article Title: The Tumor Suppressor CYLD Interacts with TRIP and Regulates Negatively Nuclear Factor ?B Activation by Tumor Necrosis Factor

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20031187

    Analysis of the interaction between CYLD and TRIP. (A) Ponceau S staining (left) shows loading of total bacterial extracts expressing GST (lane 1) and GST-TRIP (lane 2), and RIPA extracts from 293T cells either nontransfected (lane 3) or transfected with HA-CYLD (lane 4). Far Western analysis after incubating membrane with extracts from 293T cells transfected with HA-CYLD (middle) or nontransfected (right). Bound proteins were detected with anti-HA antibody. Bars indicate molecular weights of 100, 50, and 20 kD (top to bottom). (B) Ponceau S staining (left) shows loading of total bacterial extracts expressing GST-TRIP-N (lane 1, arrow) and GST-TRIP-C (lane 2, arrowhead). Far Western analysis with extracts from 293T cells transfected either with HA-CYLD (middle) or HA-lacZ (right). Bound proteins were detected with anti-HA antibody. Bars indicate molecular weights of 60 and 40 kD (top to bottom). (C) Coimmunoprecipitation experiments demonstrate interaction of CYLD with TRIP-C in 293T cells. Cells were transfected with equal amounts of FLAG-CYLD and HA-TRIP-C expression vectors. Immunoprecipitates (lanes 2 and 4) obtained with anti-FLAG antibody were analyzed by Western blots with anti-HA (top) and anti-FLAG (bottom) antibodies. Lanes 1 and 3 show input. The bars on the right side mark the molecular weights of 20 (top) and 120 kD (bottom).
    Figure Legend Snippet: Analysis of the interaction between CYLD and TRIP. (A) Ponceau S staining (left) shows loading of total bacterial extracts expressing GST (lane 1) and GST-TRIP (lane 2), and RIPA extracts from 293T cells either nontransfected (lane 3) or transfected with HA-CYLD (lane 4). Far Western analysis after incubating membrane with extracts from 293T cells transfected with HA-CYLD (middle) or nontransfected (right). Bound proteins were detected with anti-HA antibody. Bars indicate molecular weights of 100, 50, and 20 kD (top to bottom). (B) Ponceau S staining (left) shows loading of total bacterial extracts expressing GST-TRIP-N (lane 1, arrow) and GST-TRIP-C (lane 2, arrowhead). Far Western analysis with extracts from 293T cells transfected either with HA-CYLD (middle) or HA-lacZ (right). Bound proteins were detected with anti-HA antibody. Bars indicate molecular weights of 60 and 40 kD (top to bottom). (C) Coimmunoprecipitation experiments demonstrate interaction of CYLD with TRIP-C in 293T cells. Cells were transfected with equal amounts of FLAG-CYLD and HA-TRIP-C expression vectors. Immunoprecipitates (lanes 2 and 4) obtained with anti-FLAG antibody were analyzed by Western blots with anti-HA (top) and anti-FLAG (bottom) antibodies. Lanes 1 and 3 show input. The bars on the right side mark the molecular weights of 20 (top) and 120 kD (bottom).

    Techniques Used: Staining, Expressing, Transfection, Western Blot

    36) Product Images from "Apolipoprotein E4 (1-272) fragment is associated with mitochondrial proteins and affects mitochondrial function in neuronal cells"

    Article Title: Apolipoprotein E4 (1-272) fragment is associated with mitochondrial proteins and affects mitochondrial function in neuronal cells

    Journal: Molecular Neurodegeneration

    doi: 10.1186/1750-1326-4-35

    ApoE4 interacts with the subunits of mitochondrial respiratory complex IV in Neuro2a cells . Neuro2a cells were co-transfected with FLAG-apoE4 (1–272 or 1–299) plasmids and mammalian expression plasmids encoding the candidate apoE4-associated proteins. The cells were treated with 500 μl of Triton X-100 solubilization buffer and the cell lysate was incubated with anti-FLAG M2-agarose affinity resin. The immunoprecipitates were then analyzed by western blotting with an anti-COX IV 1 antibody (human COX IV 1).
    Figure Legend Snippet: ApoE4 interacts with the subunits of mitochondrial respiratory complex IV in Neuro2a cells . Neuro2a cells were co-transfected with FLAG-apoE4 (1–272 or 1–299) plasmids and mammalian expression plasmids encoding the candidate apoE4-associated proteins. The cells were treated with 500 μl of Triton X-100 solubilization buffer and the cell lysate was incubated with anti-FLAG M2-agarose affinity resin. The immunoprecipitates were then analyzed by western blotting with an anti-COX IV 1 antibody (human COX IV 1).

    Techniques Used: Transfection, Expressing, Incubation, Western Blot

    37) Product Images from "Identification and functional analysis of the pre-piRNA 3′ Trimmer in silkworms"

    Article Title: Identification and functional analysis of the pre-piRNA 3′ Trimmer in silkworms

    Journal: Cell

    doi: 10.1016/j.cell.2016.01.008

    Strategy to identify silkworm Trimmer (A) Western blot analysis of subcellular fractionated BmN4 cells. Equal amounts of total protein in each fraction were loaded. Histone H3, α-Tubulin and Tom20 were used as fraction markers of nucleus, cytoplasm and mitochondria, respectively. The crude mitochondrial (crude-mito) fraction was further separated into MAM-mito, Pure-mito, and MAM fractions. MAM, mitochondria associated membrane; Lyso/PM, lysosome and plasma membrane; ER, endoplasmic reticulum. Mitochondria enriched fractions were indicated in red. BmPapi and Trimmer are enriched in the mitochondria-containing fractions. (B) In vitro trimming assay using fractionated BmN4 cell lysate with an equal protein concentration. The mitochondria-containing fractions showed high trimming activity. (C) CHAPS-solubilized crude mitochondrial (crude-mito) fraction prepared from BmPapi-FLAG stable cells was separated by 10–35% sucrose density gradient centrifugation. In vitro trimming assay was performed using each fraction (top panel). The distribution of BmPapi and Trimmer was analyzed by Western blotting (middle panel). Total protein level in each fraction is shown (bottom panel). The distribution of BmPapi and Trimmer correlated well with the trimming activity. The fractions with high trimming activity are indicated in red. (D) CHAPS-solubilized crude mitochondrial (crude-mito) fractions were prepared from naive BmN4 or BmPapi-FLAG stable cells and subjected to immunoprecipitation with anti-FLAG antibody. In vitro trimming assay was performed using CHAPS-solubilized crude-mito fraction (IP input, left panel) or FLAG immunoprecipitates (right panel). The BmPapi-FLAG complex showed clear trimming activity. . . Mock indicates BmN4 cells transfected with dsRNAs for Renilla luciferase. Only the knockdown of BGIBMGA006602 decreased the trimming activity similarly to BmPapi knockdown. .
    Figure Legend Snippet: Strategy to identify silkworm Trimmer (A) Western blot analysis of subcellular fractionated BmN4 cells. Equal amounts of total protein in each fraction were loaded. Histone H3, α-Tubulin and Tom20 were used as fraction markers of nucleus, cytoplasm and mitochondria, respectively. The crude mitochondrial (crude-mito) fraction was further separated into MAM-mito, Pure-mito, and MAM fractions. MAM, mitochondria associated membrane; Lyso/PM, lysosome and plasma membrane; ER, endoplasmic reticulum. Mitochondria enriched fractions were indicated in red. BmPapi and Trimmer are enriched in the mitochondria-containing fractions. (B) In vitro trimming assay using fractionated BmN4 cell lysate with an equal protein concentration. The mitochondria-containing fractions showed high trimming activity. (C) CHAPS-solubilized crude mitochondrial (crude-mito) fraction prepared from BmPapi-FLAG stable cells was separated by 10–35% sucrose density gradient centrifugation. In vitro trimming assay was performed using each fraction (top panel). The distribution of BmPapi and Trimmer was analyzed by Western blotting (middle panel). Total protein level in each fraction is shown (bottom panel). The distribution of BmPapi and Trimmer correlated well with the trimming activity. The fractions with high trimming activity are indicated in red. (D) CHAPS-solubilized crude mitochondrial (crude-mito) fractions were prepared from naive BmN4 or BmPapi-FLAG stable cells and subjected to immunoprecipitation with anti-FLAG antibody. In vitro trimming assay was performed using CHAPS-solubilized crude-mito fraction (IP input, left panel) or FLAG immunoprecipitates (right panel). The BmPapi-FLAG complex showed clear trimming activity. . . Mock indicates BmN4 cells transfected with dsRNAs for Renilla luciferase. Only the knockdown of BGIBMGA006602 decreased the trimming activity similarly to BmPapi knockdown. .

    Techniques Used: Western Blot, In Vitro, Protein Concentration, Activity Assay, Gradient Centrifugation, Immunoprecipitation, Transfection, Luciferase

    Depletion of BmPapi and Trimmer causes 3′ extension and reduction of piRNAs (A) FLAG-Siwi or FLAG-BmAgo3 stable cells were transfected with dsRNAs for BmPapi and/or Trimmer. The cell extracts were subjected to immunoprecipitation by anti-FLAG antibody. The immunoprecipitates were analyzed by Western blotting (upper panel) and bound RNAs were detected by 5′ 32 P labeling after dephosphorylation (lower panel). Trimmer or BmPapi knockdown caused an extension of both Siwi- and BmAgo3-bound piRNAs. In addition, BmPapi knockdown decreased the abundance of PIWI proteins and piRNAs. (B) BmN4 cells were transfected with dsRNA for BmPapi and/or Trimmer. Total RNAs were extracted, and piRNA-1 and piRNA-2 were detected by Northern blotting. let-7 miRNA was used as a loading control. Both piRNAs were elongated by the knockdown of BmPapi and/or Trimmer. Double knockdown of BmPapi and Trimmer accumulated putative pre-piRNAs (arrowhead). (C) FLAG-Siwi or FLAG-BmAgo3 stable cells were transfected with plasmids expressing catalytically inactive (E30A) Trimmer. The cell lysates were subjected to immunoprecipitation with anti-FLAG antibody. The immunoprecipitates were analyzed by Western blotting (upper panel) and bound RNAs were detected by 5′ labeling with 32 P (lower panel). Mock indicates BmN4 cells transfected with an empty plasmid. Overexpression of catalytically inactive (E30A) Trimmer caused an extension of piRNAs. (D) BmN4 cells were transfected with plasmids expressing catalytically inactive (E30A) Trimmer. Mock indicates BmN4 cells transfected with empty plasmids. The whole cell lysates and total RNAs were extracted and analyzed by Western blotting (upper panel) or Northern blotting (lower panel). let-7 miRNA was used as a loading control for Northern blotting. Overexpression of catalytically inactive (E30A) Trimmer caused an elongation of piRNAs and accumulation of putative pre-piRNAs (arrowhead). (E, G) The length distribution of piRNAs from ~25–45 nt libraries (E) or ~35–45 nt libraries (G) that mapped to 1,811 transposable elements under double knockdown of BmPapi and Trimmer. Reads were normalized to total mapping reads. Knockdown of BmPapi and Trimmer increased the length distribution of piRNAs. ). Knockdown of BmPapi and Trimmer specifically extended at the 3′ ends of piRNAs. .
    Figure Legend Snippet: Depletion of BmPapi and Trimmer causes 3′ extension and reduction of piRNAs (A) FLAG-Siwi or FLAG-BmAgo3 stable cells were transfected with dsRNAs for BmPapi and/or Trimmer. The cell extracts were subjected to immunoprecipitation by anti-FLAG antibody. The immunoprecipitates were analyzed by Western blotting (upper panel) and bound RNAs were detected by 5′ 32 P labeling after dephosphorylation (lower panel). Trimmer or BmPapi knockdown caused an extension of both Siwi- and BmAgo3-bound piRNAs. In addition, BmPapi knockdown decreased the abundance of PIWI proteins and piRNAs. (B) BmN4 cells were transfected with dsRNA for BmPapi and/or Trimmer. Total RNAs were extracted, and piRNA-1 and piRNA-2 were detected by Northern blotting. let-7 miRNA was used as a loading control. Both piRNAs were elongated by the knockdown of BmPapi and/or Trimmer. Double knockdown of BmPapi and Trimmer accumulated putative pre-piRNAs (arrowhead). (C) FLAG-Siwi or FLAG-BmAgo3 stable cells were transfected with plasmids expressing catalytically inactive (E30A) Trimmer. The cell lysates were subjected to immunoprecipitation with anti-FLAG antibody. The immunoprecipitates were analyzed by Western blotting (upper panel) and bound RNAs were detected by 5′ labeling with 32 P (lower panel). Mock indicates BmN4 cells transfected with an empty plasmid. Overexpression of catalytically inactive (E30A) Trimmer caused an extension of piRNAs. (D) BmN4 cells were transfected with plasmids expressing catalytically inactive (E30A) Trimmer. Mock indicates BmN4 cells transfected with empty plasmids. The whole cell lysates and total RNAs were extracted and analyzed by Western blotting (upper panel) or Northern blotting (lower panel). let-7 miRNA was used as a loading control for Northern blotting. Overexpression of catalytically inactive (E30A) Trimmer caused an elongation of piRNAs and accumulation of putative pre-piRNAs (arrowhead). (E, G) The length distribution of piRNAs from ~25–45 nt libraries (E) or ~35–45 nt libraries (G) that mapped to 1,811 transposable elements under double knockdown of BmPapi and Trimmer. Reads were normalized to total mapping reads. Knockdown of BmPapi and Trimmer increased the length distribution of piRNAs. ). Knockdown of BmPapi and Trimmer specifically extended at the 3′ ends of piRNAs. .

    Techniques Used: Transfection, Immunoprecipitation, Western Blot, Labeling, De-Phosphorylation Assay, Northern Blot, Expressing, Plasmid Preparation, Over Expression

    38) Product Images from "Components of the REST/CoREST/histone deacetylase repressor complex are disrupted, modified, and translocated in HSV-1-infected cells"

    Article Title: Components of the REST/CoREST/histone deacetylase repressor complex are disrupted, modified, and translocated in HSV-1-infected cells

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

    doi: 10.1073/pnas.0502658102

    Interactions between ICP0 and the components of the CoREST/REST/HDAC complex. Nuclear lysates from mock-infected, HSV-1(F)-infected, or R7353-infected HeLa cells harvested at 2, 7, or 14 h after infection were reacted with polyclonal Ab against ICP0 ( A ) or CoREST ( B and C ). The aliquots of the lysates and immunoprecipitates were electrophoretically separated in denaturing gels and reacted with anti-CoREST, anti-HDAC1, anti-REST, or anti-ICP0 Ab as shown. Input was 10% of the total protein used for immunoprecipitation.
    Figure Legend Snippet: Interactions between ICP0 and the components of the CoREST/REST/HDAC complex. Nuclear lysates from mock-infected, HSV-1(F)-infected, or R7353-infected HeLa cells harvested at 2, 7, or 14 h after infection were reacted with polyclonal Ab against ICP0 ( A ) or CoREST ( B and C ). The aliquots of the lysates and immunoprecipitates were electrophoretically separated in denaturing gels and reacted with anti-CoREST, anti-HDAC1, anti-REST, or anti-ICP0 Ab as shown. Input was 10% of the total protein used for immunoprecipitation.

    Techniques Used: Infection, Immunoprecipitation

    39) Product Images from "Segregation of TRAF6-mediated signaling pathways clarifies its role in osteoclastogenesis"

    Article Title: Segregation of TRAF6-mediated signaling pathways clarifies its role in osteoclastogenesis

    Journal: The EMBO Journal

    doi: 10.1093/emboj/20.6.1271

    Fig. 4. TRAF6 does not associate with TRAF2 or TRAF5 in MEF cells. ( A ) TRAF6 does not heterodimerize with TRAF2 or TRAF5 when overexpressed in 293T cells. Myc-TRAF6 expression plasmid was co-transfected with control expression plasmid or expression plasmid encoding Flag-TRAF2, Flag-TRAF5 or Flag-TRAF6 into 293T cells. After 36 h, cell lysates were prepared and incubated with anti-FLAG monoclonal antibody. Co-precipitating Myc-TRAF6 was detected by immunoblot analysis with anti-Myc monoclonal antibody (top). Amounts of Myc-TRAF6 in total lysates (middle) and FLAG-TRAFs in immunocomplexes (bottom) are shown. ( B ) Endogenous TRAF6 does not associate with TRAF2 or TRAF5 upon IL-1 stimulation. MEF cells derived from wild-type mice were either unstimulated (–) or stimulated with IL-1 (20 ng/ml) for 15 min (+). Cell lysates were prepared and incubated with anti-TRAF6 antibody. Amounts of TRAF2, TRAF5, TRAF6 and IRAK in the immunoprecipitates were determined by immunoblot analysis with antibodies recognizing each protein (upper three panels). Amounts of endogenous TRAF2, TRAF5 and IRAK in the lysates were determined by immunoblotting (lower two panels).
    Figure Legend Snippet: Fig. 4. TRAF6 does not associate with TRAF2 or TRAF5 in MEF cells. ( A ) TRAF6 does not heterodimerize with TRAF2 or TRAF5 when overexpressed in 293T cells. Myc-TRAF6 expression plasmid was co-transfected with control expression plasmid or expression plasmid encoding Flag-TRAF2, Flag-TRAF5 or Flag-TRAF6 into 293T cells. After 36 h, cell lysates were prepared and incubated with anti-FLAG monoclonal antibody. Co-precipitating Myc-TRAF6 was detected by immunoblot analysis with anti-Myc monoclonal antibody (top). Amounts of Myc-TRAF6 in total lysates (middle) and FLAG-TRAFs in immunocomplexes (bottom) are shown. ( B ) Endogenous TRAF6 does not associate with TRAF2 or TRAF5 upon IL-1 stimulation. MEF cells derived from wild-type mice were either unstimulated (–) or stimulated with IL-1 (20 ng/ml) for 15 min (+). Cell lysates were prepared and incubated with anti-TRAF6 antibody. Amounts of TRAF2, TRAF5, TRAF6 and IRAK in the immunoprecipitates were determined by immunoblot analysis with antibodies recognizing each protein (upper three panels). Amounts of endogenous TRAF2, TRAF5 and IRAK in the lysates were determined by immunoblotting (lower two panels).

    Techniques Used: Expressing, Plasmid Preparation, Transfection, Incubation, Derivative Assay, Mouse Assay

    40) Product Images from "Three Yeast Proteins Related to the Human Candidate Tumor Suppressor p33ING1 Are Associated with Histone Acetyltransferase Activities"

    Article Title: Three Yeast Proteins Related to the Human Candidate Tumor Suppressor p33ING1 Are Associated with Histone Acetyltransferase Activities

    Journal: Molecular and Cellular Biology

    doi:

    Yng2-associated HAT activity is deficient in esa1 (Ts) cells. Extracts from cells containing a control plasmid pAD4.H (vector), or expressing HA-Yng2 (Yng2) or GFP-HA-Gcn5 (Gcn5) were immunoprecipitated with anti-HA. Immunoprecipitates were split, and half of each sample was examined by Western blot analysis using anti-HA antibody (top), and half was assayed for HAT activity (bottom). Strains used include FY105 (WT), gcn5 Δ, LPY3498 grown at 30°C (WT 30) or 37°C (WT 37), LPY3291 grown at 30°C ( esa1 -1 30) or 37°C ( esa1 -1 37), and LPY3500 grown at 30°C ( esa1 -2 30) or 37°C ( esa1 -2 37).
    Figure Legend Snippet: Yng2-associated HAT activity is deficient in esa1 (Ts) cells. Extracts from cells containing a control plasmid pAD4.H (vector), or expressing HA-Yng2 (Yng2) or GFP-HA-Gcn5 (Gcn5) were immunoprecipitated with anti-HA. Immunoprecipitates were split, and half of each sample was examined by Western blot analysis using anti-HA antibody (top), and half was assayed for HAT activity (bottom). Strains used include FY105 (WT), gcn5 Δ, LPY3498 grown at 30°C (WT 30) or 37°C (WT 37), LPY3291 grown at 30°C ( esa1 -1 30) or 37°C ( esa1 -1 37), and LPY3500 grown at 30°C ( esa1 -2 30) or 37°C ( esa1 -2 37).

    Techniques Used: HAT Assay, Activity Assay, Plasmid Preparation, Expressing, Immunoprecipitation, Western Blot

    Yng1, Yng2, and Pho23 are associated with HAT activities. (A) Extracts from JC1 cells expressing GFP-HA (GFP), HA-Yng2 (Yng2), HA-Ing1 (Ing1), HA-Pho23 (Pho23), HA-Yng1 (Yng1), GFP-HA-Esa1 (Esa1), or GFP-HA-Gcn5 (Gcn5) were immunoprecipitated with anti-HA (12CA5) antibody. Immunoprecipitates were split, and half of each sample was examined by Western blot analysis using anti-HA antibody (top), and half was assayed for HAT activity (bottom panels) (see Materials and Methods). The left lanes (GFP, Yng2, Ing1, Pho23, and Yng1) and right lanes (Gcn5 and Esa1) of the HAT assay were run on the same gel, but they were exposed to film for 15 and 3 days, respectively. (B) Extracts from JC1 cells expressing GFP-HA (GFP), or GFP-HA fusions with either Yng2 (Yng2), the N-terminal domain (residues 1 to 222) of Yng2 (ΔPHD), the C-terminal domain (residues 222 to 282) of Yng2 (PHD), or Yng2/1 (Yng2 residues 1 to 222 fused to Yng1 residues 155 to 219) were immunoprecipitated with anti-HA antibody. Immunoprecipitates were examined by a Western blot using anti-HA antibody (top), or HAT assays (bottom). Plasmids used to express proteins were pADGFPHA (GFP), pADHA-Yng2 (Yng2), pADHA-Ing1 (Ing1), pADHA-Pho23 (Pho23), pADHA-Yng1 (Yng1), pADGFPHA-Esa1 (Esa1), and pADGFPHA-Gcn5 (Gcn5) (in panel A) and pADGFPHA (GFP), pADGFPHA-Yng2 (Yng2), pADGFPHA-Yng2ΔPHD (ΔPHD), pADGFPHA-Yng2PHD (PHD), and pADGFPHA-Yng2/1 (Yng2/1) (in panel B). Arrows denote migration of relevant proteins. HC denotes antibody heavy chain. The panel on the lower right shows a Coomassie-stained lane from the HAT gel and the migration of histones H3, H2B, H2A, and H4.
    Figure Legend Snippet: Yng1, Yng2, and Pho23 are associated with HAT activities. (A) Extracts from JC1 cells expressing GFP-HA (GFP), HA-Yng2 (Yng2), HA-Ing1 (Ing1), HA-Pho23 (Pho23), HA-Yng1 (Yng1), GFP-HA-Esa1 (Esa1), or GFP-HA-Gcn5 (Gcn5) were immunoprecipitated with anti-HA (12CA5) antibody. Immunoprecipitates were split, and half of each sample was examined by Western blot analysis using anti-HA antibody (top), and half was assayed for HAT activity (bottom panels) (see Materials and Methods). The left lanes (GFP, Yng2, Ing1, Pho23, and Yng1) and right lanes (Gcn5 and Esa1) of the HAT assay were run on the same gel, but they were exposed to film for 15 and 3 days, respectively. (B) Extracts from JC1 cells expressing GFP-HA (GFP), or GFP-HA fusions with either Yng2 (Yng2), the N-terminal domain (residues 1 to 222) of Yng2 (ΔPHD), the C-terminal domain (residues 222 to 282) of Yng2 (PHD), or Yng2/1 (Yng2 residues 1 to 222 fused to Yng1 residues 155 to 219) were immunoprecipitated with anti-HA antibody. Immunoprecipitates were examined by a Western blot using anti-HA antibody (top), or HAT assays (bottom). Plasmids used to express proteins were pADGFPHA (GFP), pADHA-Yng2 (Yng2), pADHA-Ing1 (Ing1), pADHA-Pho23 (Pho23), pADHA-Yng1 (Yng1), pADGFPHA-Esa1 (Esa1), and pADGFPHA-Gcn5 (Gcn5) (in panel A) and pADGFPHA (GFP), pADGFPHA-Yng2 (Yng2), pADGFPHA-Yng2ΔPHD (ΔPHD), pADGFPHA-Yng2PHD (PHD), and pADGFPHA-Yng2/1 (Yng2/1) (in panel B). Arrows denote migration of relevant proteins. HC denotes antibody heavy chain. The panel on the lower right shows a Coomassie-stained lane from the HAT gel and the migration of histones H3, H2B, H2A, and H4.

    Techniques Used: HAT Assay, Expressing, Immunoprecipitation, Western Blot, Activity Assay, Migration, Staining

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    Article Snippet: .. IP was performed from isolated mouse glomeruli using anti-HA Immunoprecipitation Kit (Sigma, Cat No. IP0010). ..

    Immunoprecipitation:

    Article Title: MCP-induced protein 1 deubiquitinates TRAF proteins and negatively regulates JNK and NF-?B signaling
    Article Snippet: .. Anti-Flag M2 affinity gel, anti-HA immunoprecipitation kit, protein G immunoprecipitation kit, LPS (Escherichia coli 026:B6-derived), human recombinant IFN-γ, IL-1β, and TNF, NEM, and other chemical reagents were purchased from Sigma-Aldrich. .. Ubiquitin chains, ubiquitin-AFC, ubiquitin-agarose, IsoT, ubiquitin aldehyde, and MG132 were purchased from Boston Biochem.

    Article Title: Involvement of Tetraspanin C189 in Cell-to-Cell Spreading of the Dengue Virus in C6/36 Cells
    Article Snippet: .. Immunoprecipitation (IP) An anti-HA immunoprecipitation kit (Sigma, MO, USA) was utilized for this experiment. .. In brief, C6/36 cells were cultured in a 6-well plate until about 80% of a monolayer was formed.

    Article Title: Extracellular Interactions between Hepatitis C Virus and Secreted Apolipoprotein E
    Article Snippet: .. Anti-β-actin (AC15) and HA monoclonal antibodies (HA-7), an anti-HA immunoprecipitation kit, an HA peptide, and a horseradish peroxidase-conjugated goat anti-mouse IgG were purchased from Sigma-Aldrich. .. Alexa Fluor 488-labeled donkey anti-goat IgG and Alexa Fluor 594-labeled donkey anti-mouse IgG were purchased from Thermo Fisher Scientific.

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    other:

    Article Title: CDK phosphorylation of Xenopus laevis M18BP1 promotes its metaphase centromere localization
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    Lysis:

    Article Title: Arginine GlcNAcylation of Rab small GTPases by the pathogen Salmonella Typhimurium
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    Recombinant:

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    SDS Page:

    Article Title: CDK phosphorylation of Xenopus laevis M18BP1 promotes its metaphase centromere localization
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    Article Title: The MKK-Dependent Phosphorylation of p38α Is Augmented by Arginine Methylation on Arg49/Arg149 during Erythroid Differentiation
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    Millipore brca1
    Adriamycin and cis -platinum cause loss of telomeric <t>BRCA1</t> in T47D cells. Cells were sham-treated or exposed to ADR or CDDP for the indicated times and harvested for telomeric ChIP assays to detect TRF1 and BRCA1 ( A and D ). As negative controls, ChIP assays were performed using normal goat IgG (control for TRF1 IP) or mouse IgG (control for BRCA1 IP). Dot blots were quantified using densitometry, normalized to the input, and expressed relative to sham-treated controls, as means ± S.E. of three independent experiments (*, p
    Brca1, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore icp8
    Knockdown of BAF reduces the size and numbers of viral RCs, but heterochromatin is excluded from the RCs. HFF cells were transfected with NT control or BAF-specific siRNA and infected with HSV-1 at an MOI of 10. At 8 hpi, the cells were fixed and processed for indirect immunofluorescence assays with antibodies specific for BAF (red in top row), <t>ICP8</t> (red in bottom row), and H3K9me3 (green). Arrows indicate ICP8-stained RCs.
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    Millipore anti cre
    Knockdown of BAF reduces the size and numbers of viral RCs, but heterochromatin is excluded from the RCs. HFF cells were transfected with NT control or BAF-specific siRNA and infected with HSV-1 at an MOI of 10. At 8 hpi, the cells were fixed and processed for indirect immunofluorescence assays with antibodies specific for BAF (red in top row), <t>ICP8</t> (red in bottom row), and H3K9me3 (green). Arrows indicate ICP8-stained RCs.
    Anti Cre, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Adriamycin and cis -platinum cause loss of telomeric BRCA1 in T47D cells. Cells were sham-treated or exposed to ADR or CDDP for the indicated times and harvested for telomeric ChIP assays to detect TRF1 and BRCA1 ( A and D ). As negative controls, ChIP assays were performed using normal goat IgG (control for TRF1 IP) or mouse IgG (control for BRCA1 IP). Dot blots were quantified using densitometry, normalized to the input, and expressed relative to sham-treated controls, as means ± S.E. of three independent experiments (*, p

    Journal: The Journal of Biological Chemistry

    Article Title: BRCA1 Localization to the Telomere and Its Loss from the Telomere in Response to DNA Damage *

    doi: 10.1074/jbc.M109.025825

    Figure Lengend Snippet: Adriamycin and cis -platinum cause loss of telomeric BRCA1 in T47D cells. Cells were sham-treated or exposed to ADR or CDDP for the indicated times and harvested for telomeric ChIP assays to detect TRF1 and BRCA1 ( A and D ). As negative controls, ChIP assays were performed using normal goat IgG (control for TRF1 IP) or mouse IgG (control for BRCA1 IP). Dot blots were quantified using densitometry, normalized to the input, and expressed relative to sham-treated controls, as means ± S.E. of three independent experiments (*, p

    Article Snippet: As expected, there were no changes in telomeric BRCA1 or TRF1, total cellular hTERT or BRCA1, or immunoprecipitatable BRCA1 protein.

    Techniques: Chromatin Immunoprecipitation

    BRCA1 regulates 3′ G-strand overhang length; hybridization protection assay. A , standard curves of luminescence (relative units) versus genomic DNA input were obtained using an AE-labeled telomeric probe ( left ) or an AE-labeled AluDNA probe ( right ). Data are shown for DNA treated with or without Exo I, which removes single-stranded DNA. B–G , cells were treated with the indicated siRNAs and/or transfected overnight with wild-type ( wt ) BRCA1 or empty pcDNA3 vector, and genomic DNA (5 μg) was assayed to determine the ratio of luminescence (arbitrary units ( a.u. )) obtained using the telomeric and Alu probes. Controls using Exo I and, in some cases, negative controls ( no DNA ) are provided. C , a Western blot to document overexpression of BRCA1 in cells transfected with wild-type BRCA1. H , the telomeric probe signal for genomic DNA (5 μg) treated with T7 exonuclease (which digests duplex DNA, but not single-stranded DNA, in a 5′ to 3′ direction) for different time intervals. All data are means ± S.E. of three independent experiments.

    Journal: The Journal of Biological Chemistry

    Article Title: BRCA1 Localization to the Telomere and Its Loss from the Telomere in Response to DNA Damage *

    doi: 10.1074/jbc.M109.025825

    Figure Lengend Snippet: BRCA1 regulates 3′ G-strand overhang length; hybridization protection assay. A , standard curves of luminescence (relative units) versus genomic DNA input were obtained using an AE-labeled telomeric probe ( left ) or an AE-labeled AluDNA probe ( right ). Data are shown for DNA treated with or without Exo I, which removes single-stranded DNA. B–G , cells were treated with the indicated siRNAs and/or transfected overnight with wild-type ( wt ) BRCA1 or empty pcDNA3 vector, and genomic DNA (5 μg) was assayed to determine the ratio of luminescence (arbitrary units ( a.u. )) obtained using the telomeric and Alu probes. Controls using Exo I and, in some cases, negative controls ( no DNA ) are provided. C , a Western blot to document overexpression of BRCA1 in cells transfected with wild-type BRCA1. H , the telomeric probe signal for genomic DNA (5 μg) treated with T7 exonuclease (which digests duplex DNA, but not single-stranded DNA, in a 5′ to 3′ direction) for different time intervals. All data are means ± S.E. of three independent experiments.

    Article Snippet: As expected, there were no changes in telomeric BRCA1 or TRF1, total cellular hTERT or BRCA1, or immunoprecipitatable BRCA1 protein.

    Techniques: Hybridization, Labeling, Transfection, Plasmid Preparation, Western Blot, Over Expression

    Teli-FISH of BRCA1 colocalization with telomeric probe. Subconfluent proliferating T47D cells on slides were fixed and hybridized to a Cy3-labeled telomere-specific PNA probe. The slides were immunostained with BRCA1, TRF1, or TRF2 primary antibody and secondary antibody conjugated to Alexa Fluor dye and processed for confocal imaging. Images are shown of DAPI stain ( blue ), PNA probe ( red ), BRCA1 ( green ), TRF1 ( green ), or TRF2 ( green ). Quantification of colocalization of BRCA1, TRF1, or TRF2 with the PNA probe ( yellow granules in merged images ) was carried out using Metamorph version 6.2 software ( n = 28) or by manual counting of the percentage of total telomeric granules ( yellow + red ) that is colocalized ( yellow ) with BRCA1, TRF1, or TRF2 ( n = 3 cells).

    Journal: The Journal of Biological Chemistry

    Article Title: BRCA1 Localization to the Telomere and Its Loss from the Telomere in Response to DNA Damage *

    doi: 10.1074/jbc.M109.025825

    Figure Lengend Snippet: Teli-FISH of BRCA1 colocalization with telomeric probe. Subconfluent proliferating T47D cells on slides were fixed and hybridized to a Cy3-labeled telomere-specific PNA probe. The slides were immunostained with BRCA1, TRF1, or TRF2 primary antibody and secondary antibody conjugated to Alexa Fluor dye and processed for confocal imaging. Images are shown of DAPI stain ( blue ), PNA probe ( red ), BRCA1 ( green ), TRF1 ( green ), or TRF2 ( green ). Quantification of colocalization of BRCA1, TRF1, or TRF2 with the PNA probe ( yellow granules in merged images ) was carried out using Metamorph version 6.2 software ( n = 28) or by manual counting of the percentage of total telomeric granules ( yellow + red ) that is colocalized ( yellow ) with BRCA1, TRF1, or TRF2 ( n = 3 cells).

    Article Snippet: As expected, there were no changes in telomeric BRCA1 or TRF1, total cellular hTERT or BRCA1, or immunoprecipitatable BRCA1 protein.

    Techniques: Fluorescence In Situ Hybridization, Labeling, Imaging, Staining, Software

    BRCA1 association with TRF1 and TRF2 is mediated by DNA. Cells were subjected to reciprocal IP-Western blotting for BRCA1/TRF1 ( A and C ) or BRCA1/TRF2 ( B and D ). In C and D , IPs were carried out using lysates pretreated with or without DNase I (100 units/ml for 30 min at 37 °C). For each IP, a control IP using the same quantity of normal IgG was performed, and an input lane is provided corresponding to 10% of the amount of protein used for IP. E , Western blots of unprecipitated lysates treated with or without DNase I; F , electrophoresis of lysates pretreated with or without DNase I on a 1% agarose gel.

    Journal: The Journal of Biological Chemistry

    Article Title: BRCA1 Localization to the Telomere and Its Loss from the Telomere in Response to DNA Damage *

    doi: 10.1074/jbc.M109.025825

    Figure Lengend Snippet: BRCA1 association with TRF1 and TRF2 is mediated by DNA. Cells were subjected to reciprocal IP-Western blotting for BRCA1/TRF1 ( A and C ) or BRCA1/TRF2 ( B and D ). In C and D , IPs were carried out using lysates pretreated with or without DNase I (100 units/ml for 30 min at 37 °C). For each IP, a control IP using the same quantity of normal IgG was performed, and an input lane is provided corresponding to 10% of the amount of protein used for IP. E , Western blots of unprecipitated lysates treated with or without DNase I; F , electrophoresis of lysates pretreated with or without DNase I on a 1% agarose gel.

    Article Snippet: As expected, there were no changes in telomeric BRCA1 or TRF1, total cellular hTERT or BRCA1, or immunoprecipitatable BRCA1 protein.

    Techniques: Western Blot, Electrophoresis, Agarose Gel Electrophoresis

    Knockdown of BAF reduces the size and numbers of viral RCs, but heterochromatin is excluded from the RCs. HFF cells were transfected with NT control or BAF-specific siRNA and infected with HSV-1 at an MOI of 10. At 8 hpi, the cells were fixed and processed for indirect immunofluorescence assays with antibodies specific for BAF (red in top row), ICP8 (red in bottom row), and H3K9me3 (green). Arrows indicate ICP8-stained RCs.

    Journal: mBio

    Article Title: Barrier-to-Autointegration Factor 1 (BAF/BANF1) Promotes Association of the SETD1A Histone Methyltransferase with Herpes Simplex Virus Immediate-Early Gene Promoters

    doi: 10.1128/mBio.00345-15

    Figure Lengend Snippet: Knockdown of BAF reduces the size and numbers of viral RCs, but heterochromatin is excluded from the RCs. HFF cells were transfected with NT control or BAF-specific siRNA and infected with HSV-1 at an MOI of 10. At 8 hpi, the cells were fixed and processed for indirect immunofluorescence assays with antibodies specific for BAF (red in top row), ICP8 (red in bottom row), and H3K9me3 (green). Arrows indicate ICP8-stained RCs.

    Article Snippet: The supernatant lysates were subjected to immunoprecipitati on with an antibody specific for FLAG (M2, Sigma) or ICP8 ( , ) or with control normal rabbit IgG (Millipore).

    Techniques: Transfection, Infection, Immunofluorescence, Staining

    Effect of BAF on viral transcript levels. HFF cells were transfected with NT control or BAF-specific siRNA and infected with HSV-1 at an MOI of 10. (A) Knockdown efficiency was confirmed by quantitative RT-PCR. (B to D) Cells were harvested at the postinfection times indicated, total RNAs were prepared, and IE ( ICP4 [B] and ICP27 [C]) and E ( ICP8 [D]) gene mRNA levels were measured by quantitative RT-PCR. The mRNA levels were normalized to 18S rRNA. Results shown are means and standard deviations from three independent experiments.

    Journal: mBio

    Article Title: Barrier-to-Autointegration Factor 1 (BAF/BANF1) Promotes Association of the SETD1A Histone Methyltransferase with Herpes Simplex Virus Immediate-Early Gene Promoters

    doi: 10.1128/mBio.00345-15

    Figure Lengend Snippet: Effect of BAF on viral transcript levels. HFF cells were transfected with NT control or BAF-specific siRNA and infected with HSV-1 at an MOI of 10. (A) Knockdown efficiency was confirmed by quantitative RT-PCR. (B to D) Cells were harvested at the postinfection times indicated, total RNAs were prepared, and IE ( ICP4 [B] and ICP27 [C]) and E ( ICP8 [D]) gene mRNA levels were measured by quantitative RT-PCR. The mRNA levels were normalized to 18S rRNA. Results shown are means and standard deviations from three independent experiments.

    Article Snippet: The supernatant lysates were subjected to immunoprecipitati on with an antibody specific for FLAG (M2, Sigma) or ICP8 ( , ) or with control normal rabbit IgG (Millipore).

    Techniques: Transfection, Infection, Quantitative RT-PCR

    BAF localizes to viral RCs. (A) HFF cells were infected with HSV-1 at an MOI of 10, and an indirect immunofluorescence assay was performed with antibodies specific for BAF and ICP8 at 8 hpi. Images were deconvoluted by the inverse filter algorithm in the AxioVision 4.8 image acquisition software. (B) HeLa cell expressing FLAG-BAF were infected with HSV-1 DG1 at an MOI of 50, harvested at 4 hpi, and immunoprecipitated (IP) with antibodies specific for FLAG or ICP8. ICP4, ICP8, VP16-GFP, and FLAG-BAF were detected with antibodies specific for ICP4, ICP8, GFP, and FLAG.

    Journal: mBio

    Article Title: Barrier-to-Autointegration Factor 1 (BAF/BANF1) Promotes Association of the SETD1A Histone Methyltransferase with Herpes Simplex Virus Immediate-Early Gene Promoters

    doi: 10.1128/mBio.00345-15

    Figure Lengend Snippet: BAF localizes to viral RCs. (A) HFF cells were infected with HSV-1 at an MOI of 10, and an indirect immunofluorescence assay was performed with antibodies specific for BAF and ICP8 at 8 hpi. Images were deconvoluted by the inverse filter algorithm in the AxioVision 4.8 image acquisition software. (B) HeLa cell expressing FLAG-BAF were infected with HSV-1 DG1 at an MOI of 50, harvested at 4 hpi, and immunoprecipitated (IP) with antibodies specific for FLAG or ICP8. ICP4, ICP8, VP16-GFP, and FLAG-BAF were detected with antibodies specific for ICP4, ICP8, GFP, and FLAG.

    Article Snippet: The supernatant lysates were subjected to immunoprecipitati on with an antibody specific for FLAG (M2, Sigma) or ICP8 ( , ) or with control normal rabbit IgG (Millipore).

    Techniques: Infection, Immunofluorescence, Software, Expressing, Immunoprecipitation