Peptide Competition.
Lysates prepared from HeLa cells left untreated (1) or treated with anisomycin (2-5) were resolved by SDS-PAGE on a 14% polyacrylamide gel and transferred to PVDF. Membranes were blocked with a 5% BSA-TBST buffer for one hour at room  temperature, and incubated with ab12864 for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 2), the non-phosphopeptide corresponding to the immunogen (3), a generic phosphoserine-containing peptide (4), or, the phosphopeptide immunogen (5). After washing, membranes were incubated with a goat anti-rabbit IgG HRP conjugate secondary antibody and bands were detected using the Pierce SuperSignal^TM method. The data show that only the peptide corresponding to RPS6 blocks the signal, verifying the specificity of the antibody. Peptide Competition. Lysates prepared from HeLa cells left untreated (1) or treated with anisomycin (2-5) were resolved by SDS-PAGE on a 14% po