Peptide Competition and Phosphatase Treatment: Lysates prepared from Jurkat cells stimulated with PMA 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  phosphatase (5), blocked with a 5% low-fat milk-TBST buffer for one hour at room temperature, and incubated with ab5813 antibody for two hours at room temperature in a 3% low-fat milk-TBST buffer, following prior incubation with: no peptide (1), 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 SuperSignalTM method. The data show that the phosphopeptide corresponding to PKC iota [pT555] blocks the antibody signal. The peptide corresponding to PKC zeta [pT560] blocks the antibody signal and the peptides corresponding to PKC isoforms beta 1 [pT642] and gamma [pÔ655] partially block the antibody signal (data not shown), suggesting cross-reactivity of the antibody with these sites. The antibody signal was not blocked by the corresponding peptides of any other PKC isoforms. The data also show that phosphatase stripping eliminates the signal, verifying that the antibody is phospho-specific.

Peptide Competition and Phosphatase Treatment: Lysates prepared from Jurkat cells stimulated with PMA 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 phosphatase (5), blocked with a 5% low-fat milk-TBST buffer for one hour at room temperature, and incubated with ab5813 antibody for two hours at room temperature in a 3% low-fat milk-TBST buffer, following prior incubation with: no peptide (1), 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 SuperSignalTM method. The data show that the phosphopeptide corresponding to PKC iota [pT555] blocks the antibody signal. The peptide corresponding to PKC zeta [pT560] blocks the antibody signal and the peptides corresponding to PKC isoforms beta 1 [pT642] and gamma [pÔ655] partially block the antibody signal (data not shown), suggesting cross-reactivity of the antibody with these sites. The antibody signal was not blocked by the corresponding peptides of any other PKC isoforms. The data also show that phosphatase stripping eliminates the signal, verifying that the antibody is phospho-specific.