Fig. 2. Temporal activation of Cdc42 and Rac1 by thrombin and S1P. Confluent monolayers of serum-starved HUVECs were treated for the times indicated with 10 nM thrombin or 0.5 µM SIP. Western blot analyses of active CRIB-bound Rac1 and Cdc42 are shown in the upper panels, and total proteins are shown in the lower panels. The results shown are representative of two independent experiments in which the effects of thrombin and S1P were directly compared. Fold stimulation of GTP-bound Rho proteins was determined following normalization against total GTPases.