FIGURE 5.

Loss of BCAR3 expression disrupts cell spreading and adhesion-mediated Cas phosphorylation. A and B, BT549 cells depleted for BCAR3 exhibit decreased cell spreading on fibronectin. BT549 cells were transfected with nontargeting or BCAR3-specific siRNA duplexes and grown for 72 h. Cells were left in suspension for 90 min and then plated on fibronectin-coated coverslips in complete growth media with 10% fetal bovine serum for the indicated lengths of time in normal growth media. A coverslip from cycling, adherent cells was also examined. Mean cell area from three independent experiments was determined by using ImageJ software. Bars indicate standard deviation; asterisk indicates significant difference from the mean at ≥95% confidence interval. C, BT549 cells depleted for BCAR3 exhibit decreased adhesion-mediated Cas phosphorylation. BT549 cells were transfected and treated as described above. Cas immune complexes were generated from lysate and immunoblotted with Tyr(P) and Cas antibodies (top two panels). Whole cell lysate was also immunoblotted with antibodies specific to BCAR3 (bottom panel). Data shown are representative of four independent experiments. Within all panels, exposure time is the same; the white vertical line denotes noncontiguous sample lanes. IP, immunoprecipitation.