Peptide Competition. Recombinant eIF2Bepsilon treated with lambda phosphatase (1) or GSK-3beta (2-5) were added to background extracts and resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF. The membrane was blocked with a 5% BSA-TBST buffer overnight at 4°C, then incubated with the eIF2Bepsilon (pS539) 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 phosphoserine-containing peptide (4), or the phosphopeptide immunogen (5). After washing, the membrane was incubated with goat F (ab’)2 anti-rabbit IgG alkaline phosphatase (Product # ALI4405) and signals were detected using the Tropix WesternStar™ method. The data show that only the peptide corresponding to eIF2Bepsilon (pS539) blocks the antibody signal, demonstrating the specificity of the antibody. The data also show that phosphatase stripping eliminates the signal, verifying that the antibody is phospho-specific. The recombinant eIF2Bepsilon was kindly provided by Dr. Scot R. Kimball, Penn State University.