Peptide Competition: Cell extracts prepared from NIH 3T3 cells expressing EGFR were starved for 30 hours, then stimulated for 10 minutes with 30 ng/mL EGF (+), or left unstimulated (-), then resolved by SDS-PAGE on a 6% Tris-glycine gel, and transferred to nitrocellulose. The membranes were then incubated with 0.50 µg/mL ab5638 antibody, following prior incubation in the absence of the peptide immunogen (1, 2), presence of the peptide immunogen (3, 4), or the non-phosphopeptide corresponding to the EGFR phosphopeptide (5, 6). After washing, membranes were incubated with goat F(ab’)2 anti-rabbit IgG alkaline phosphatase and bands were detected using the Tropix WesternStar detection method. The data show that only the phosphopeptide corresponding to this site blocks the antibody signal, demonstrating the specificity of the ab5638 antibody for this phosphorylated residue. The data also show the activation of the EGFR after stimulation with EGF. Peptide Competitio