Activated Rac inhibits thrombin signaling to Rho. (A) PAE cells transfected with pCEFL-GFP as a control or with the activated RacV12 mutant and pCEFL-GFP together (RacV12) were starved overnight on poly-D-lysine coated glass cover-slips, treated with thrombin 5 U/ml for indicated times, fixed, and stained with TRITC-phalloidin. Arrows point to transfected cells as judged by co-transfection with pCEFL-GFP. (B) HEK293T cells were transfected with pCEFL-GFP as a control or with the activated RacV12 mutant and pCEFL-GFP together with RacV12 (RacV12), cultured overnight, starved for 4 h, and treated with thrombin for indicated times. Fresh lysates were used for Rho-pulldown assays with GST-Rhotekin to detect relative amounts of Rho-GTP. Total Rho levels were detected by western blot in total cell lysates as a loading control.