Vav2 is required for flow-induced Rac1 GTP loading, whereas Tiam1 regulates spatial activation of Rac1. (A) VE-RC or VE-KO ECs were plated on fibronectin-coated slides and shear stress applied for the indicated times. Cell lysates were subjected to SDS-PAGE followed by immunoblotting with phospho- and total Vav2 antibodies. Three experiments were performed and numbers under the gel panels represent the ratio of pVav2/total Vav2 normalized to t = 0. (B) PE-RC or PE-KO ECs were plated on fibronectin-coated slides and shear stress applied for the indicated times. Total Vav2 protein was immunoprecipitated from cell lysates and subjected to SDS-PAGE, followed by immunoblotting with phospho- and total Vav2 antibodies. Three experiments were performed and numbers under the gel panels represent the ratio of pVav2/total Vav2 normalized to t = 0 (top). Shear stress was applied to HUVECs on fibronectin-coated slides and shear stress was applied in the presence of DMSO or the Src family kinase inihibitor, SU6656. Total Vav2 was immunoprecipitated from cell lysates and immune complexes were subjected to SDS-PAGE. Western blots were performed to assess phospho-Vav2 levels (bottom). (C) ECs were transfected with control siRNA, siVav2#1 or siTiam1#1. 72 h after transfections, cells were plated on fibronectin-coated slides and shear stress applied for the indicated times. Rac1-GTP pull-down assays were performed to assess the Rac1-GTP loading. (D) HUVECs were transfected with indicated siRNAs and subsequently sheared or kept as static control. FRET images were obtained and cells scored for total FRET and downstream FRET. Values are mean ± SEM, n = 3 and >100 cells were scored per condition. *, P < 0.05; **, P < 0.01. Bar, 10 µm.