Peptide Competition and Stimulation.,.Extracts prepared from CSF-1-stimulated (1-4, 6) or unstimulated (5) PAE cells transfected with a chimeric CSF-1/VEGFR2 receptor (see Positive Controls Used section above for full description) 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 0.50 ug/mL VEGFR2 [pY1214] antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 5, 6), the nonphosphopeptide corresponding to the immunogen (2), a generic phosphotyrosine containing peptide (3), or, the phosphopeptide immunogen (4). 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 [pY1214] blocks the antibody signal, and the stimulation of the phospho-signal after stimulating ligand is added (CSF-1 for the chimeric receptor, compare lanes 5 and 6), thereby demonstrating the specificity of the antibody.