Figure 5. Reciprocal role of Abi1-Iso2 in Crk-mediated Abl transactivation and in Crk-Abl1b axis-mediated cell motility and invasion.

A. HS683 stable cell line overexpressing Abi1-Iso2 shows significant reduction in Crk Y251 and Abl Y245 phosphorylation in time dependent manner. B. Wound-healing assay: Cell migration of HS683 stable cells was assessed by imaging the rate of wound healing by serum starved, hEGF stimulated cells in triplicate at 6 and 12hrs time-points. C. Percentage wound healing was calculated and represented in the bar graph (n = 3) (P < 0.05). D. Real-time xCELLigence assay for cell invasion: Cells were grown to 80% confluency, serum starved and seeded in triplicates in matrigel coated upper chambers of CIM plates. 10% FBS containing media was used as chemoattractant in the lower chamber of CIM plate. Change in Cell index was measured every 15min for 36-48 hours. E. Abi1−/− and Abi1 fl/fl MEFs were serum-starved overnight and stimulated with 100ng/ml of hEGF. Enhanced CrkY251 and AblY245 phosphorylation was observed in Abi1−/− MEFs as compared with Abi1 fl/fl cells. F. Densitometry of phosphoprotein bands were calculated, normalized by ImageJ and represented in bar graphs. G.-H. Crk and Abl kinase complexes that form with or without EGF treatment in Abi1−/− MEFs were investigated by immunoprecipitation of Crk after diluent or hEGF stimulation. I.-J. Reverse immunoprecipitation by Abl antibody and immunoblotting with Crk antibody was performed. K.-L. Wound healing/scratch assay: Abi1−/− MEFs, were transiently transfected as indicated. 48hrs post-transfection, cells were serum starved for 16hrs and assayed for wound healing as described (see methods). M. Microimpedance-based cell spreading assay: 10,000 cells transfected as described in panel K were seeded on fibronectin coated E-plate 16 and assessed by real-time cell spreading assay every minute by xCELLigence RTCA DP. Data are represented as mean ± SEM, and n = 3 for Panel C and F. See also Supplementary Figure S6.