Cell extracts prepared from NIH3T3 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. Membranes were incubated with 0.50 µg/mL  ab5650 antibody, following prior incubation in the absence (lanes 1& 2), or presence of the peptide immunogen (lanes 3 & 4), or the nonphosphopeptide corresponding to the EGFR phosphopeptide (lanes 5 & 6). After washing, membranes were incubated with goat F(ab’)2 antirabbit 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 ab5650 antibody for this phosphorylated residue. Cell extracts prepared from NIH3T3 cells expressing EGFR were starved for 30 hours, then stimulate