Fig. 1. CrkII protects p130^cas from dephosphorylation. (A) 293-EBNA cells were transfected with either pRK-VSV-CrkII (2 µg) or pRK-GFP-PTP1B (2 µg) expression plasmids, or co-transfected with pRK-VSV-CrkII and pRK-GFP-PTP1B. Cell lysates were immunoprecipitated with anti-p130^cas (IP: p130cas), anti-paxillin (IP: paxillin) or anti-FAK (IP: FAK) antibodies, and then immunoblotted with anti-phosphotyrosine (Blot: pTyr), anti-p130^cas (Blot: p130cas), anti-paxillin (Blot: paxillin) or anti-FAK (Blot: FAK) antibodies. The whole-cell lysates (WCL) were immunoblotted with anti-GFP (Blot: GFP) or anti-VSV (Blot: VSV) antibodies. The numerical values indicate relative ratios as a percentage of the control for each band of phosphoprotein/total protein after scanning and analysis using NIH Image software. (B) pRK-GFP-PTP1B (2 µg) and various quantities of pcDNA-CrkII expression plasmids as indicated were co-transfected in 293-EBNA cells. Cell lysates were immunoprecipitated with anti-p130^cas (IP: p130cas) and then immunoblotted with anti-phosphotyrosine (Blot: pTyr), anti-p130^cas (Blot: p130cas) or anti-Crk (Blot: Crk) antibodies. The whole-cell lysates (WCL) were immunoblotted with anti-GFP (Blot: GFP) or anti-Crk (Blot: Crk) antibodies. The numbers indicate relative ratios as a percentage of controls for phosphoprotein/total protein or for CrkII bound to p130^cas compared with the control.