Peptide Competition (left panel) Lysates prepared from SK-BR-3 cells were resolved by SDS-PAGE on an 18% polyacrylamide gel and transferred to PVDF. Membranes were blocked with a 5% BSA-TBST buffer for one hour at room temperature and incubated with PED/PEA-15 [pS116] antibody for one hour at room temperature in 3% BSA-TBST buffer, following prior incubation with: no peptide (1), the nonphosphopeptide corresponding to the immunogen (2), a generic phosphoserine-containing peptide (3), or, the phosphopeptide immunogen (4). 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. Mutant experiments (right panel) Lysates were prepared from PMA-stimulated Cos7 cells transfected with HA-tagged wild-type PED/PEA-15 (A), His-tagged S104A mutant PED/PEA-15 (B), and Histagged S116A mutant PED/PEA-15 (C). Proteins were resolved by SDS-PAGE, transfered to PVDF, and blotted with PED/PEA-15 [pS116] antibody as above.