TPeptide Competition and Phosphatase Treatment: Lysates prepared from NIH3T3 cells stimulated with PDGF were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were either left untreated (1-5) or treated with lambda phosphatase (6), blocked with a 5% BSA-TBST buffer for one hour at room temperature, and incubated with ab10564 antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 2, 6), 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 goat F(ab’)2 anti-rabbit IgG HRP conjugate and bands were detected using the chemilumiescence. The data show that the peptide corresponding to ab10564 blocks the antibody signal. The data also show that phosphatase stripping eliminates the signal, verifying that the antibody is phospho-specific. TPeptide Competition and

Immunohistochemistry (PFA perfusion fixed frozen section) staining of CREB (phospho S129 + S133) in the mouse cortex. Samples were fixed with paraformaldehyde and ab10564 was used at 1/1000 for 18 hours at 25ºC. An Alexa Fluor®568 conjugated donkey anti-Rabbit secondary was used at 1/1000.

See Abreview