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. 2010 Aug 16;31(10):1293–1302. doi: 10.1038/aps.2010.96

Figure 3.

Figure 3

Acetylation of KLF4 by p300 enhances its binding to the mfn-2 promoter. (A) Identification of anti-Ac-lys antibody. VSMCs were treated with 10 μmol/L ATRA for 2 h, cell lysates were immunoprecipitated using an irrelevant antibody (nonimmune IgG) or the antibodies against KLF4 and acetylated lysine, the precipitates were detected via Western blotting using anti-KLF4 or anti-acetylated lysine antibodies. (B) ATRA induced KLF4 acetylation. VSMCs were treated with 10 μmol/L ATRA for the indicated times. Cell lysates were immunoprecipitated with anti-KLF4 antibody, and acetylated KLF4 was detected via Western blotting using anti-Ac-lys antibody. Blots for total KLF4 are also shown. (C) ATRA increased the interaction of KLF4 with p300. VSMCs were treated with ATRA for 1 h. Cell lysates were immunoprecipitated with anti-KLF4 or anti-p300 antibodies as indicated (IP). The precipitates were analyzed by Western blotting (IB) with anti-p300 and anti-KLF4 antibodies, respectively. (D) KLF4 was acetylated by p300 in vitro. KLF4 or its acetylation-deficient mutants (300 ng) and p300 (100 ng) were incubated with acetyl-CoA for 30 min at 30 °C, and reaction products were separated by SDS-PAGE, and then acetylated KLF4 was determined by Western blotting using anti-Ac-lys antibody. Blots for total KLF4 are also shown. (E) The interaction of p300 with KLF4 and its acetylation-deficient mutants. The lysates of VSMCs treated with or without ATRA were incubated with GST, GST-KLF4, GST-KLF4 (K225R), GST-KLF4 (K229R), or GST-KLF4 (K225/229R). After pull down with GST beads, p300 was detected via Western blotting using anti-p300 antibody, and KLF4 was detected with anti-GST antibody. (F) Acetylation of KLF4 enhanced the mfn-2 promoter activity. Luciferase assay was performed in A293 cells transfected with the mfn-2 promoter-reporter plasmid (containing nucleotides −441 to +15 of the mfn-2 promoter), along with different combinations of expression plasmids for KLF4 or its acetylation-deficient mutant (K225/229R), p300, and deacetylase HDAC2. The bars represent the means±SE from three independent experiments. bP<0.05 vs the control.