Article Title: Identification of Novel Protein Lysine Acetyltransferases in Escherichia coli
Figure Lengend Snippet: Mutation of conserved catalytic amino acids prevents RimI-, PhnO-, YjaB-, and YiaC-dependent acetylation. The gutted strain (BW25113 Δ pta yfiQ acs cobB ) was transformed with the pCA24n vector control, pCA24n carrying the wild-type allele for each putative KAT, or mutant alleles for each putative KAT with alanine substitutions of the indicated residues. The resulting strains were grown in TB7 supplemented with 0.4% glucose, 100 μM IPTG, and 25 μg/ml chloramphenicol for 8 h. Crude lysates harvested after 4 h were analyzed for expression of the KAT proteins. Whole-cell lysates harvested after 8 h were analyzed for acetylation. Coomassie blue-stained SDS-PAGE gels (A and C) served as loading controls for anti-His (B) and anti-acetyllysine (D) Western blot analysis.
Article Snippet: Acetylated peptides were enriched using 1/4 tube of the anti-acetyllysine antibody-bead-conjugated PTMScan Acetyl-Lysine Motif [Ac-K] kit (Cell Signaling Technologies) for each of the 1-mg protein lysate samples according to the manufacturer’s instructions.
Techniques: Mutagenesis, Transformation Assay, Plasmid Preparation, Expressing, Staining, SDS Page, Western Blot