Subcellular localization of RhoA-GTP, Rac1-GTP, and Cdc42-GTP, and of GFP-tagged Rac1 and RhoA. (A) ECs (i-iv) and ECs carrying Rac1-GFP (v-viii) were starved and stimulated with vehicle (i,v) or with Ang-1 (20 ng/mL) for 2 (ii,vi), 5 (iii,vii), and 120 (iv,viii) minutes. Panels i-iv are merged images of F-actin (green) and GST-PBD (red). Arrows indicate lamellipodia. (B) ECs (i-iv) and ECs carrying RhoA-GFP (v-viii) were starved and stimulated with vehicle (i,v) or with Ang-1 (20 ng/mL) for 2 (ii,vi), 5 (iii,vii), and 120 (iv,viii) minutes. Panels i-iv are merged images of F-actin (green) and GST-RBD (red). The arrowhead and the white arrows indicate F-actin cables and ruffles, respectively. (C) Starved, subconfluent ECs were stimulated for 2 hours (or 2 minutes, not shown) with vehicle or Ang-1 (20 ng/mL) and underwent pull-down and immunolocalization assays with GST-WBD. (D) Pull-down assays for Rac1, RhoA, and Cdc42 from lysates of fixed and permeabilized ECs. The results of densitometric analysis of Rac1-GTP, RhoA-GTP, and Cdc42-GTP where normalized to total Rac1, RhoA, and Cdc42 in whole cell lysates and expressed to the basal value of unstimulated cells (mean ± SDs of 3 independent experiments). Bars indicate 10 μm. Figures are representative of at least 3 experiments.