Fig. 6. CrkII-Y221F accelerates cell motility on fibronectin. (A) HT1080 cells were co-transfected with 0.5 µg of pHA262pur, 0.5 µg of pRK-GFP, and 0.5 µg or 2 µg of pcDNA-CrkII-WT and pcDNA-CrkII-Y221F, or 2 µg of CrkII-W169L, psSR-DN-p130^cas or control plasmids, and transfectants were selected by puromycin for 36 hours. After puromycin selection, cells were replated on 35 mm glass dishes coated with 10 µg/ml fibronectin, and cultured overnight in serum-free DMEM containing 1 mg/ml BSA. Cell movements were monitored for 3 hours by time-lapse video microscopy. Error bars indicate s.d. for at least 30 cells per condition (^*P<0.001 versus controls). Parallel cultures of these cells were homogenized and subjected to immunoblotting with anti-Crk (WCL Blot: Crk) antibody. (B) Phase contrast microscopy of HT1080 cells co-transfected with 0.5 µg of pHA262pur, pRK-GFP, and 0.5 µg (low) or 2 µg (high) of pcDNA-CrkII-WT, CrkII-Y221F or control plasmids 12 hours after replating onto fibronectin. Magnification, x200.