Extracts of 3T3L1 cells stimulated with 100 nM insulin for 10 minutes 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 and either left untreated (1-4) or treated with lambda (λ) phosphatase (5), then incubated with the GSK-3 α [pY279] / β [pY216] antibody for two hours at room temperature in a 1% BSATBST buffer, following its prior incubation with: the phosphopeptide immunogen (1), no peptide (2), the non-hosphopeptide corresponding to the phosphopeptide immunogen (3), or a generic phosphotyrosine-containing peptide (4). After washing, the membrane was incubated with goat F(ab’) 2 anti-rabbit IgG HRP conjugate, and signals were detected. The data show that only the phosphopeptide corresponding to GSK-3α [pY279] /β[pY216] blocks the antibody signal, demonstrating the specificity of the antibody. The data also show that phosphatase stripping eliminates the signal, further verifying that the antibody is phospho-specific.