Human ProS inhibits VEGF-A–induced VEGFR2 activation at SHP2 sensitive sites. (A-B) Western blotting analysis of VEGFR2 phosphorylation after cultured EC exposure to the indicated factors. Subconfluent EC cultures were preincubated in a growth factor-depleted medium with either human ProS at the indicated concentrations or with vehicle for 15 minutes and then were exposed for 5 minutes to 20 ng/mL VEGF-A. Cells were exposed to the SHP2 inhibitor NSC 87877 at 100μM for 3 hours or vehicle before ProS. Cell cultures were then lysed, and equivalent amounts of protein form each sample were resolved by SDS-PAGE, transferred to a PVDF membrane, and probed with anti–phospho-VEGFR2 Tyr⁹⁹⁶, Tyr¹¹⁷⁵, or anti-VEGFR2 antibodies. The intensity of bands was quantified and is represented in panel C as a percentage of control of the ratio of phosphorylated VEGFR2 over VEGFR2 ± SD. Data were obtained from 3 independent experiments (each in triplicates) expressed as percentages of control ± SD. ***P < .001. ns indicates not significant.