Fig. 4.

β-Arrestins are critical for APC-induced Rac1 activation but not for thrombin-induced RhoA activation. (A and B) Endothelial cells transfected with 100 nM nonspecific (ns) or β-arrestin siRNAs were stimulated with 10 nM thrombin (Th) or 20 nM APC for 5 min or 180 min at 37 °C. Cells were lysed. Equivalent amounts of cell lysates were incubated with GST-PBD or GST-RBD, and the amount of activated Rac1 or RhoA was determined by immunoblotting. Total cell lysates were immunoblotted with an anti-Rac1 or anti-RhoA antibody as a control. Data (mean ± SEM) are expressed as the fold increase over untreated control (Ctrl) of three independent experiments. The differences between Rac1 or RhoA activity observed in agonist-treated cells versus untreated control cells were significant (*P < 0.05; **P < 0.01).