Extracts of primary chicken embryo fibroblasts (CEF) mock transfected (1) or transfected with a vector containing FAK (2-6), plated on fibronectin and treated with 50 μM vanadate for 16 hours were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF. The membrane was blocked with a 5% BSA-TBST buffer for one hour at room temperature, then incubated with the FAK [pY576] antibody for two hours at room temperature in a 1% BSA-TBST buffer, following prior incubation with: no peptide (1, 2), the non-phosphopeptide corresponding to the phosphopeptide immunogen (3), a generic phosphotyrosine-containing peptide (4), a phosphopeptide derived from the corresponding region of Pyk2 (5), or the phosphopeptide immunogen (6). After washing, the membrane was incubated with goat F(ab')2 anti-rabbit IgG HRP-conjugate, and signals were detected using the Pierce SuperSignal™ method.