FIGURE 8:

Increased phosphorylation of p130^CAS and Rac1 activation in Y925E-FAK cells. (A) Increased Rac1 activity in Y925E-FAK cells compared with wild-type FAK and Y925F-FAK cells. Cells were serum starved for 24 h before stimulation by fibronectin for 30 min. Rac activation was measured by G-LISA, and absorbance was read at 490 nm. Graph combines data from four experiments performed independently. (B) Cell extracts were immunoprecipitated using anti-Dock180 antibody and blotted for Rac1. Membranes were stripped and verified for Dock180 expression. (C) Equal expression levels of p130^CAS in wild-type FAK, Y925F-FAK, and Y925E-FAK cells. Phosphorylated p130^CAS was determined by immunoprecipitating phosphorylated proteins with an anti-phosphotyrosine antibody followed by blotting with a p130^CAS antibody. Mean phospho-p130^CAS data (mean ± SEM; n = 3 independent experiments). (D). Confocal images from fixed cells; YCam-tagged wild-type or mutant FAK (green) and immunostaining for phospho-p130^CAS (red); merged images (right). Scale bar, 10 μm.