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. 2003 Oct 1;22(19):5036–5046. doi: 10.1093/emboj/cdg503

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Copyright © 2003 European Molecular Biology Organization

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graphic file with name cdg503f7a.jpg — Fig. 7. SOCS-proteins promote polyubiquitination of FAK. (A) COS-7 cells were transiently transfected with HA-FAK (0.5 µg) alone or with wild-type forms of Myc-SOCS-1 or SOCS-3 or their SOCS box-deletion mutants (SOCSΔSB) (1.0 µg). Twenty-four hours after transfection the cells were serum-starved overnight and pretreated with or without β-lactacystin (25 µM) for 4 h prior to pervanadate treatment (50 µM) for 1 h. FAK was immunoprecipitated with an anti-HA antibody, followed by a dissociation and reprecipitation with the same antibody. FAK immunoprecipitates were analyzed by immunoblotting with antibodies against ubiquitin and HA. Total cell lysates were subjected to immunoblotting with anti-Myc antibody to confirm SOCS-protein expression levels. (B) In vitro ubiquitination reactions on immunoprecipitated wild-type FAK or FAK-Y397F mutant were performed as described in the Materials and methods in the presence or absence of cell lysates containing the indicated SOCS proteins. The reaction products were separated on SDS–PAGE, transferred onto PVDF membrane, and the blots were probed with anti-ubiquitin antibody (upper panel). Equal loading of the various SOCS proteins was verified by anti-Myc immunoblotting of total cell lysates (lower panel). (C) HA-tagged wild-type FAK was generated by a coupled in vitro transcription/translation system. An in vitro ubiquitination assay was performed on the Sepharose-purified FAK as in (B). (D) NIH 3T3 cells were transfected with 0.5 µg of plasmid coding for ubiquitin with or without 1.0 µg of SOCS-3ΔSB. Twenty-four hours after transfection, the cells were serum-starved overnight, detached, stimulated or not with PDGF, and either kept in suspension or replaced on FN for 1 or 4 h in the presence of 50 µM pervanadate and 25 µM proteasome inhibitor MG132. Cell lysates were subjected to immunoprecipitation with anti-FAK antibody followed by immunoblotting with antibodies against ubiquitin, FAK, pTyr, SOCS-3 and Myc (upper panels). An isotype-matched IgG was used as a control to demonstrate that anti-FAK antibodies do not unspecifically precipitate SOCS-3 (middle panel). Total cell lysates were subjected to immunoblot with anti-SOCS-3, anti-Myc and anti-tubulin antibodies (bottom three panels).

graphic file with name cdg503f7b.jpg — Fig. 7. SOCS-proteins promote polyubiquitination of FAK. (A) COS-7 cells were transiently transfected with HA-FAK (0.5 µg) alone or with wild-type forms of Myc-SOCS-1 or SOCS-3 or their SOCS box-deletion mutants (SOCSΔSB) (1.0 µg). Twenty-four hours after transfection the cells were serum-starved overnight and pretreated with or without β-lactacystin (25 µM) for 4 h prior to pervanadate treatment (50 µM) for 1 h. FAK was immunoprecipitated with an anti-HA antibody, followed by a dissociation and reprecipitation with the same antibody. FAK immunoprecipitates were analyzed by immunoblotting with antibodies against ubiquitin and HA. Total cell lysates were subjected to immunoblotting with anti-Myc antibody to confirm SOCS-protein expression levels. (B) In vitro ubiquitination reactions on immunoprecipitated wild-type FAK or FAK-Y397F mutant were performed as described in the Materials and methods in the presence or absence of cell lysates containing the indicated SOCS proteins. The reaction products were separated on SDS–PAGE, transferred onto PVDF membrane, and the blots were probed with anti-ubiquitin antibody (upper panel). Equal loading of the various SOCS proteins was verified by anti-Myc immunoblotting of total cell lysates (lower panel). (C) HA-tagged wild-type FAK was generated by a coupled in vitro transcription/translation system. An in vitro ubiquitination assay was performed on the Sepharose-purified FAK as in (B). (D) NIH 3T3 cells were transfected with 0.5 µg of plasmid coding for ubiquitin with or without 1.0 µg of SOCS-3ΔSB. Twenty-four hours after transfection, the cells were serum-starved overnight, detached, stimulated or not with PDGF, and either kept in suspension or replaced on FN for 1 or 4 h in the presence of 50 µM pervanadate and 25 µM proteasome inhibitor MG132. Cell lysates were subjected to immunoprecipitation with anti-FAK antibody followed by immunoblotting with antibodies against ubiquitin, FAK, pTyr, SOCS-3 and Myc (upper panels). An isotype-matched IgG was used as a control to demonstrate that anti-FAK antibodies do not unspecifically precipitate SOCS-3 (middle panel). Total cell lysates were subjected to immunoblot with anti-SOCS-3, anti-Myc and anti-tubulin antibodies (bottom three panels).