Peptide Competition:
Extracts prepared from HEK293 cells transiently transfected with plasmids expressing ERK5 kinase domain (ERK5kin) and constitutively activated MEK5D-D were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were blocked with a 5% BSA TBST buffer overnight at 4oC, then were incubated with the ab5686 antibody for two hours at room temperature in a 3% BSATBST buffer, following prior incubation with: no peptide (1), the non-phosphopeptide corresponding to the immunogen (2), a generic phosphothreonine-containing peptide (3), a generic phosphotyrosine-containing peptide (4), the phosphopeptide derived from the corresponding region of ERK1&2 (5), or, the phosphopeptide immunogen (6). After washing, membranes were incubated with goat F(ab’ 2 anti-rabbit IgG alkaline phosphatase conjugate and bands were detected using the Tropix WesternStarTM detection method. The data show that while there is some cross-reactivity with ERK1&