Decreased VEGF–induced VEGFR2 phosphorylation in Rap1-deficient ECs. (A) A time-course of VEGF-induced VEGFR2 phosphorylation in WT and Rap1b^−/− ECs. Maximal VEGF-induced VEGFR2 phosphorylation occurring at 5 minutes in WT ECs is inhibited in Rap1b^−/− ECs. (B-C) Analysis of VEGF- induced VEGFR2 total tyrosine phosphorylation (B) and VEGFR2 pY1054/1059 phosphorylation (C) in WT ECs and in WT ECs upon Rap1b or Rap1a siRNA knockdown, as indicated. Rap1 deficiency in WT ECs leads to reduced VEGFR2 pY1054/1059 phosphorylation (C) and total VEGFR2 tyrosine phosphorylation (B), similar to that observed in Rap1b^−/− ECs in panel A. Efficiency of siRNA silencing was tested by Western blot for Rap1b (B) or total Rap1 (C). To normalize for protein content, a sample of lysates before IP was probed for actin (A-C bottom blots). Western blots shown are of typical experiments. Graphs represent mean fold induction in Rap1-deficient ECs normalized to WT EC values (“WT-V5”; n = 4 independent experiments; error bars are SEM).