Up-regulation and Antibody-Peptide Competition.
Peptide Competition. Human recombinant Tau untreated (lane 1) or treated with GSK-3β (1 µg per µg Tau) for 45 minutes (lanes 2-5) was added to background extracts, 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 then incubated with the Tau [pS199] antibody in a 3% BSA-TBST buffer for two hours at room temperature, 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 HRP conjugate (Cat. no. ALI4404) and signals were detected using the Pierce SuperSignal™ method. The data show that only the peptide corresponding to Tau [pS199] blocks the antibody signal, demonstrating the specificity of the antibody. The data also show the induction of Tau [pS199] phosphorylation by the addition of GSK-3β.