Figure 2.

A synthetic acetylation substrate is acetylated in vivo. A, synthetic fusion constructs used for acetylation analyses. B, GFP fusions were purified from strains expressing the constructs in A using an antibody against GFP. Eluates were analyzed via SDS-PAGE and probed either with anti-acetyllysine (αAcK) that recognizes the consensus or αGFP following Western blotting. Diluted forms ($\raisebox{1ex}{$1$}\!\left/ \!\raisebox{-1ex}{$25$}\right.$) of immunoprecipitated protein samples were loaded for GFP detection. C, the indicated constructs were purified from strains treated with 0, 5, or 20 mm NAM for 30 min and analyzed as in B. D, indicated constructs were purified from the strains shown using the GFP Trap reagent prior to NuPAGE analysis, staining by colloidal Coomassie, and analysis of excised bands by MS. E, acetylated peptides detected by MS analysis. Acetylations on these sites were detected in two of three independent purifications. Also see Fig. S2.