Activated Rac requires Pak1 binding for inhibition of thrombin signaling to Rho. (A) PAE cells transfected with pCEFL-GFP as a control or with RacV12, RacV12C40, RacV12Δins mutants together with pCEFL-GFP as indicated were starved overnight on poly-D-lysine coated glass cover-slips, placed in serum-free media (control) or treated with thrombin (5 U/ml) 30 min, fixed and stained with TRITC-labelled phalloidin. Transfected cells were detected by GFP flourescence. (B) HEK293T cells were transfected with pCEFL-GFP as a control or with the RacV12 mutants shown in (A) together with pCEFL-GFP, cultured overnight, starved 4 h, treated with thrombin (5 U/ml) for 1 min, and lysed. 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.