Fig. 2. PTP1B directly dephosphorylates CrkII. (A) An in-blot tyrosine phosphatase assay was performed as described in Materials and Methods. Immunoprecipitates using anti-VSV antibody (IP: VSV) from lysates of 293-EBNA cells co-transfected with VSV-FAK and VSV-CrkII were subjected to SDS-PAGE and electroblotting. The membranes were incubated with recombinant human PTP1B (20 U/ml) at 37°C for 30 minutes, then immunoblotted with anti-phosphotyrosine (Blot: pTyr) or anti-VSV antibodies (Blot: VSV). (B) 293-EBNA cells were co-transfected with either 0.5 µg of pcDNA CrkII-WT or CrkII-Y221F with or without pRK-GFP-PTP1B (2 µg), and then homogenized in NP-40 buffer. The cell lysates were immunoprecipitated with anti-Crk (IP: Crk) polyclonal antibody, and then immunoblotted with anti-phosphotyrosine (Blot: pTyr) or anti-Crk (Blot: Crk) monoclonal antibodies. The whole-cell lysates (WCL) were immunoblotted with anti-GFP antibody (Blot: GFP). (C) Immunoprecipitates using anti-CrkII antibody (IP: CrkII) from lysates of 293-EBNA cells co-transfected with pcDNA-CrkII and either pRK-GFP-PTP1B or GFP-PTP1B-C215S were subjected to SDS-PAGE and immunoblotting with anti-phosphotyrosine (Blot: pTyr) or anti-CrkII (Blot: CrkII) antibodies. The whole-cell lysates were immunoblotted with anti-GFP-antibody (Blot: GFP). (D) 293-EBNA cells co-transfected with either pcDNA-CrkII-WT or CrkII-Y221F were homogenized, and the lysates were immunoprecipitated with either anti-p130^cas (IP: p130cas) or anti-paxillin (IP: paxillin) antibodies, and then immunoblotted with anti-Crk (Blot: Crk), anti-p130^cas (Blot: p130cas), or anti-paxillin (Blot: paxillin) antibodies.