Fig. 1. Temporal activation of Rho A by thrombin and S1P. Confluent monolayers of HUVECs were cultivated as indicated in Materials and Methods and, after overnight starvation, were treated with thrombin (10 nM) or S1P (0.5 µM) for the times indicated. The amount of RBD-bound and total RhoA in cell extracts was determined by western analysis. A minor band, not regulated by agonists, was often detected above total RhoA, which likely corresponds to a geranyl-geranylated form of the protein. The results shown are representative of three independent experiments in which thrombin and S1P effects were directly compared. Fold stimulation of GTP-bound Rho was determined following normalization against total Rho.