Lysates prepared from 3T3-L1 cells were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were either left untreated (1-4) or treated with lambda (λ) phosphatase (5), blocked with a 5% BSA-TBST buffer for two hours at room temperature, and incubated with PKA regIIβ[pS114] antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 5), the non-phosphopeptide corresponding to the immunogen (2), a generic phosphothreonine-containing peptide (3), or, the phosphopeptide immunogen (4). After washing, membranes were incubated with goat F(ab') 2 anti-rabbit IgG HRP conjugate and bands were detected using the Pierce SuperSignal method. The data show that the peptide corresponding to PKA regIIβ[pS114] blocks the antibody signal. The data also show that phosphatase stripping eliminates the signal, verifying that the antibody is phospho-specific.