Inhibition of VEGF-induced VEGFR2 phosphorylation by TSR is mediated through SHP-1. MVEC transfected with control siRNA or siRNA directed against SHP-1 or SHP-2 (A) or against Fyn or Syk (B) were exposed to 10 nmol/L TSR in low-serum medium for 4 hours and then to 50 ng/mL VEGF for 5 minutes. Phosphorylated VEGFR2 in these samples were analyzed by western blot with pTyr1175 antibody. Blots were reprobed for VEGFR2 and phosphorylation levels expressed as a ratio of phosphorylated to total VEGFR2 (n = 3). The images above the bar graphs are representative western blots showing the efficiency of protein knock-down 48 hours after transfection. Anti-VEGFR2 or anti-IgG control immunoprecipitates of MVEC (C) or MVEC silenced with respective siRNA (D) were incubated with the phosphatase substrate pNPP for 1 hour at room temperature, and phosphatase activity was detected by absorbance at 405 nm.