Peptide Competition – Mutant Analysis Lysates prepared from COS cells overexpressing human wild-type PAG3 alone (1) cotransfected with human wild-type PAG3 and mouse Pyk2 (2-5), or mutant [Y724F/Y763F] PAG3 and mouse Pyk2 (6) were resolved by SDS-PAGE on an 8% polyacrylamide gel and transferred to PVDF. Membranes were blocked with a 5% BSA-TBST buffer for one hour at room temperature, then incubated with PAG3 [pY724] antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 2, 6), the nonphosphopeptide corresponding to the immunogen (3), a generic phosphotyrosinecontaining peptide (4), or, the phosphopeptide immunogen (5). After washing, membranes were incubated with goat F(ab’)2 anti-rabbit IgG HRP conjugate in 3% BSA-TBST buffer, and bands were detected using the Pierce SuperSignal method. The data show that only the peptide corresponding to PAG3 [pY724] blocks the signal, and the signal is absent in the mutant extracts, demonstrating the specificity of the antibody. Cell extracts were kindly provided by Dr. Hisataka Sabe, Osaka Bioscience Institute, Osaka, Japan.