Peptide Competition and Phosphatase Treatment: Lysates prepared from A431 cells left unstimulated (1) or stimulated with EGF (2-6) were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were either not treated (1-5) or treated with lambda phosphatase (6), blocked with a 5% BSA-TBST buffer for one hour at room temperature, and incubated with ab5482 antibody for two hours at room temperature in a 3% BSATBST buffer, following prior incubation with: no peptide (1, 2, 6), the nonphosphopeptide corresponding to the immunogen (3), a generic phosphoserine containing peptide (4), or, the phosphopeptide immunogen (5), After washing, membranes were incubated with goat F(ab’)2 anti-rabbit IgG HRP conjugate and bands were detected using the Pierce SuperSignal method. The data show that the peptide corresponding to ab5482 blocks the antibody signal. The data also shows that phosphatase stripping eliminates the signal, verifying that the antibody is phospho-specific.