Peptide Competition and Phosphatase Treatment:
Lysates prepared from Jurkat cells left untreated (1) or treated with hydrogen peroxide (2-6) were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were either left untreated (1-5) or treated with lambda (λ) phosphatase (6), blocked with a 5% BS A-TBST buffer for one hour at room temperature, and incubated with ZAP-70 [pY292] antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 6), the non-phosphopeptide corresponding to the immunogen (2), a generic phosphotyrosine-containing peptide (4) or, the phosphopeptide immunogen (5). After washing, membranes were incubated with goat F(ab')2 anti-rabbit IgG HRP conjugate (Cat. # ALI4404), and bands were detected using the Pierce SuperSignal™ method.