FIG 2.

K88 can be acetylated by AcP. (A) The specificity of anti-K88Ac antibody. Wild-type PhoP protein (WT) and site-specific acetylated K88Ac, K102Ac, and K201Ac proteins were resolved on 12% SDS-PAGE and probed with anti-K88Ac antibody and anti-PhoP antibody. (B) The acetylation level of PhoP K88 in macrophages. After infecting RAW264.7 cells for 24 h, the intracellular bacteria were harvested for IP assay. The immunoprecipitated PhoP protein by anti-Flag antibody was analyzed by Western blotting with anti-K88Ac antibody. Representative results from three independent experiments of Western blots are shown. The relative K88 acetylation level over PhoP was quantified. Error bars indicate ± SDs of triplicate experiments; **, P < 0.01. Student’s t test. (C) The acetylation of PhoP K88 after knockout of pat or cobB. PhoP proteins from the wild-type strain and mutation strain of S. Typhimurium 14208s were detected by Western blotting and probed with anti-K88Ac antibody, and anti-PhoP antibody was used as a loading control. Representative results from three independent experiments of Western blots are shown. The relative K88 acetylation level over PhoP was quantified. Error bars indicate ± SDs of triplicate experiments. *, P < 0.05. ns, no significance. Student’s t test. (D) The acetylation level of PhoP K88 isolated from strains cultured in LB medium supplemented with glucose. The wild-type strain and pta or ackA mutation strains of S. Typhimurium 14208s were cultured in LB medium with or without 0.8% (wt/vol) glucose added and then harvested for IP assay. The immunoprecipitated PhoP protein by anti-Flag antibody was analyzed by Western blotting with anti-K88Ac antibody. Representative results from three independent experiments of Western blots are shown. The relative K88 acetylation level over PhoP was quantified (top panel). Error bars indicate ± SDs of triplicate experiments; *, P < 0.05. Student’s t test. (E) PhoP K88 was acetylated by AcP in vitro. Purified His-tagged PhoP was incubated with AcP as an acetyl group donor for different durations, and then samples were detected by Western blot analysis with anti-pan acetyl lysine antibody (α-Pan Ac), which recognized acetylated lysine residues and anti-K88Ac antibody. Representative results from three independent experiments of Western blots are shown. The relative pan acetylation or K88 acetylation level over PhoP was quantified. Error bars indicate ± SDs of triplicate experiments; *, P < 0.05; **, P < 0.01. ns, no significance. Student’s t test.