Peptide Competition and Stimulation.,.Extracts prepared from NIH3T3 cells transfected with human VEGFR2 and left unstimulated (1) or stimulated with VEGF (2-5) were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were blocked with a 5% BSA-TBST buffer overnight at 4oC, then were incubated with 0.40 ug/mL VEGFR2 [pY951] antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 2), the non-phosphopeptide corresponding to the immunogen (3), a generic phospho-tyrosine containing peptide (4), or, the phosphopeptide immunogen (5). After washing, membranes were incubated with goat F(ab’)2 anti-rabbit IgG alkaline phosphatase and bands were detected using the Tropix WesternStar method. The data show that the signal is strongly induced by VEGF treatment and that only the peptide corresponding to VEGFR2 [pY951] blocks the antibody signal, thereby demonstrating the specificity of the antibody