Fig. 3. Fibronectin promotes dephosphorylation of CrkII. (A) 293-EBNA and HT1080 cells were serum-starved for 6 hours or 12 hours, respectively. The cells were trypsinized, kept in suspension for 20 minutes, re-plated onto culture dishes coated with 10 µg/ml fibronectin for 10, 30 or 120 minutes, and then homogenized. The 293-EBNA lysates were immunoprecipitated with anti-p130^cas (IP: p130cas) or anti-Crk (IP: Crk) antibodies, and then immunoblotted with anti-phosphotyrosine (Blot: pTyr), anti-p130^cas (Blot: p130cas) or anti-Crk (Blot: Crk) antibodies. The whole-cell lysates (WCL) from HT1080 cells were immunoblotted with anti-Crk (Blot: Crk) antibody. (B) HT1080 cells were serum-starved for 12 hours, trypsinized, kept in suspension for 20 minutes, then replated onto culture dishes coated with 10 µg/ml fibronectin for 30 or 120 minutes. The cells were homogenized and immunoprecipitated with anti-p130^cas (IP: p130cas) or anti-Crk (IP: Crk) antibodies, and then immunoblotted with anti-phosphotyrosine (Blot: pTyr), anti-p130^cas (Blot: p130cas) or anti-Crk (Blot: Crk) antibodies. (C) HT1080 cells in 100 mm dishes were co-transfected with 1 µg of pHA262pur and 2 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 replated onto culture dishes coated with 10 µg/ml fibronectin for 2 hours. The cells were homogenized and immunoprecipitated with anti-p130^cas (IP: p130cas) antibody, and then immunoblotted with anti-phosphotyrosine (Blot: pTyr) or anti-p130^cas (Blot: p130cas) antibodies. The whole-cell lysates (WCL) were immunoblotted with anti-PTP1B (Blot: PTP1B) or anti-Crk (Blot: Crk) antibodies.