A) Newly developed MEK1(AcK175) antibody detects acetylated Flag-MEK1, but not Flag-MEK1(K175R) in HEK 293T cells. The MEK1 (AcK175) polyclonal antibody was developed by conjugating keyhole limpet hemocyanin antigen to a 12 amino acid peptide corresponding to human MEK1 acetylated at position K175. Antisera from immunized rabbits was passed through a column coupled with the unacetylated peptide to remove antibody that reacted with non-acetylated MEK1. B) Ectopic expression of either SIRT1 or SIRT2 dampens MEK1(K175) acetylation. C) HeLa cells display enriched endogenous acetylated MEK1(K175) in the nucleus following EGF stimulation. Overnight serum-deprived HeLa cells were left untreated or treated with EGF, fixed with paraformaldehyde, and analyzed by IF. Endogenous MEK1 was detected using pan MEK1 antibody or MEK1(AcK175) antibody. Pre-incubation of the MEK1(AcK175) antibody with acetylated MEK1 peptide (2 µg), but not the non-acetylated peptide (2 µg), blocked detection of acetylated MEK1 (bottom panels). D) K175 and K362 are important for regulating MEK1-dependent phosphorylation of ERK. Glucose and serum deprived HEK 293T cells expressing Flag-MEK1 wild-type (WT), or site-directed mutants (K175R, K175Q, K362R, K362Q) were left untreated or stimulated with EGF. Immunoprecipitated MEK1 proteins were used in in vitro kinase assays as described in Figure 1E. E) Immunoprecipitated acetyl-mimic MEK1(K175/362Q) displays elevated kinase activity. For D and E, the fold change in ERK phosphorylation was determined as described in Figure 1. Similar fold changes in ERK phosphorylation were obtained in three individual experiments. F) The acetyl-mimic MEK1(K175/362Q) effectively stimulates ELK transcriptional activity. Glucose and serum deprived HEK 293T cells transiently expressing the Gal4-luciferase reporter, Gal4-ELK, and Flag tagged wild-type (WT), or MEK1 mutants were either left alone (No Add) or treated with U0126, followed by EGF stimulation for 8 hrs prior to harvesting. Data are a calculated mean ± S.D. P-values were calculated relative to No Add or EGF stimulated wild-type MEK1 (WT), *p <0.05, N = 3, (ns) not significant.