Peptide Competition and Stimulation.,.Extracts prepared from PAE cells transfected with a chimeric receptor (see Positive Controls section above for full description) were left unstimulated (1, 2) or stimulated with CSF (3-8), then 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 a VEGFR2 pan antibody (1, 3) or 0.50 ug/mL VEGFR2 [pYpY1054/1059] antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (2, 4, 5), the non-phosphopeptide corresponding to the immunogen (6), a generic phosphotyrosine containing peptide (7), or, the phosphopeptide immunogen (8). 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 only the peptide corresponding to VEGFR2 [pYpY1054/1059] blocks the antibody signal, and the stimulation of the phospho signal after VEGF is added (compare lanes 2 and 4), thereby demonstrating the specificity of the antibody. Note that the chimeric CSF-1-VEGFR2 is ~190 kDa.