Fig. 7. Co-expression of CrkII and PTP1B promotes cell migration. (A) HT1080 cells in 100 mm dishes were co-transfected with 1 µg of pHA262pur and 0.5 µg of pcDNA-CrkII with 0.5 µg or 1.0 µg of pRK-GFP-PTP1B, and transfectants were selected by puromycin for 36 hours. After puromycin selection, cells were serum-starved for 12 hours and then replated on culture dishes coated with 10 µg/ml fibronectin for 2 hours. The cells were homogenized and immunoprecipitated with anti-Crk (IP: Crk), anti-p130^cas (IP: p130cas), or anti-paxillin (IP: paxillin) antibodies, and then immunoblotted with anti-phosphotyrosine (Blot: pTyr), anti-Crk (Blot: Crk), anti-p130^cas (Blot: p130cas), or anti-paxillin (Blot: paxillin) antibodies. The whole-cell lysates (WCL) were immunoblotted with anti-PTP1B (Blot: PTP1B) antibody. (B) HT1080 cells were co-transfected with 0.5 µg of pHA262pur with 0.3 µg of pcDNA-CrkII-WT or pcDNA-CrkII-Y221F, and 0.5 µg of pRK-GFP-PTP1B, and transfectants were selected by puromycin for 36 hours. Cell migration was measured using 48-well chemotaxis chambers as described in Materials and Methods. Error bars indicate s.d. for at least three experiments (^*P<0.05 versus controls).