Inhibition of signaling from integrin β₃ to VEGFR2 in Rap1b-deficient ECs. (A-C). Analysis of VEGF-induced VEGFR2 phosphorylation in WT and Rap1b^−/− ECs on inhibition of integrin β₃ with function blocking antibody (A) or by siRNA silencing (B); and in integrin β₃–knockout ECs on Rap1b siRNA knockdown (C). VEGFR2 immunoprecipitates were blotted with a phosphotyrosine-specific antibody (top blots). To normalize for protein content, a sample of lysates pre-IP was probed for actin (bottom blots). (D) Silencing integrin β₁ in WT or Rap1b^−/− ECs does not inhibit VEGF-induced VEGFR2 phosphorylation. Efficiency of siRNA silencing was tested by Western blot for integrin β₃, Rap1b and β₁ (B-D, respectively). (E) VEGF does not induce VEGFR2 phosphorylation in WT or Rap1b^−/− ECs plated on collagen or poly-L-lysine. Blots shown are of typical experiments and graphs represent quantification of normalized percent VEGFR2 phosphorylation (shaded bars) relative to WT (A) or control siRNA-transfected (B-C) controls (filled bars). Values shown are mean of 4 independent experiments, error bars represent SEM. (F) C-Raf-1 phosphorylation is not changed in Rap1b^−/− ECs. Serine 338 phosphorylation of C-Raf-1 was assessed in quiescent or VEGF-stimulated (5 minutes, 40 ng/mL) WT and Rap1b^−/− ECs. Total C-Raf-1 and actin blots of the same gel were performed to ensure equal protein loading.