Ang-1 regulates thrombin-induced actin stress fiber formation, Rho activity, and Ca⁺⁺ flux. (A) EC monolayers were unstimulated (i), stimulated with thrombin (0.2 U/mL) alone for 10 minutes (ii), or pretreated with Ang-1 (0.1 μg/mL) for 30 minutes followed by thrombin (iii). Cells were stained using rhodamine-phalloidin for detection of F-actin. (B) Cells were either unstimulated (Nil), stimulated with thrombin alone (T), or pretreated with Ang-1 and then stimulated with thrombin (Ang-1 + T) and analyzed for active Rho (top panel). Total Rho in the cell lysates is shown in the bottom panel. (C) Cells were loaded with fura 2 either in the absence or presence of Ang-1 (0.1 μg/mL) for 60 minutes. Cells were then treated with thrombin (0.2 U/mL) and fluorescence was measured. The pooled results, expressed as fluorescence ratio, are given for 100 cells from each of 3 experiments.