p190RhoGEF facilitates integrin-associated RhoA regulation, FA formation, and motility of normal MEFs. (A) Stable p190RhoGEF knockdown. FAK^+/+ MEFs were infected with lentiviral Scr or p190RhoGEF shRNA and sorted for GFP expression and lysates were analyzed by p190RhoGEF, FAK, actin, and GFP blotting. (B) p190RhoGEF knockdown limits RhoA activation upon FN adhesion. GTP-bound RhoA was determined by GST-RBD pulldown assays from lysates of suspended and FN-replated FAK^+/+ parental, FAK^+/+ Scr shRNA, and FAK^+/+ p190RhoGEF shRNA MEFs, followed by blotting for total RhoA levels. (C) Quantitation of FN-stimulated RhoA-GTP binding from assays shown in B. Values are means ± SD of two independent experiments and are plotted relative to RhoA-GTP levels in suspended FAK^+/+ MEFs. (D) p190RhoGEF knockdown limits FA formation but not MEF spreading on FN. FAK^+/+ Scr and FAK^+/+ p190RhoGEF shRNA MEFs were plated onto 1-μg/ml-FN–coated coverslips for 60 min and costained with antibodies to paxillin and Alexa Fluor 350 phalloidin for F-actin. Bar, 30 μm. (E) Decreased FA formation upon p190RhoGEF knockdown. Box-and-whisker plots of paxillin-positive–stained points within FAK^+/+ Scr shRNA or FAK^+/+ p190RhoGEF shRNA MEFs plated on FN for 60 min (n = 15 cells per point). (F) 10-μg/ml-FN–stimulated FAK^+/+ (parental), FAK^+/+ Scr, and FAK^+/+ p190RhoGEF shRNA MEF Boyden chamber motility over 4 h. Means ± SD were determined from three experiments and are a percentage of FAK^+/+ control (set to 100).