Extracts prepared from NIH3T3 cells left untreated (lane 1) or treated (lanes 2-5) with PDGF were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to nitrocellulose. Membranes were blocked with a 5% BSA TBST buffer overnight at 4^oC, then were incubated with 0.50 µg/mL MEK 1 & 2 [pS222] antibody, following prior incubation with: no peptide (1, 2) the nonphosphopeptide corresponding to the immunogen (3), a dual phosphopeptide corresponding to MEK 1 & 2 [pSpS218/222] (4), the phosphopeptide immunogen (5). After washing, membranes were incubated with goat F(ab’)2 anti-rabbit IgG alkaline phosphatase and signals were detected using the Tropix WesternStar method. The data show that the peptides corresponding to MEK 1 & 2 [pS222] and MEK 1 & 2 [pSpS218/222] block the antibody signal, but the corresponding nonphospho-peptide does not, thereby demonstrating t