Figure 5

Lysine 182 of Nkx2.5 is deacetylated by SIRT1.

(a) Luciferase assay showing that the mutation of lysine 182 reduces the transcriptional activity of Nkx2.5. pGL3-ANF-Luc plasmid was co-transfected with WT and mutant Nkx2.5 into HEK293A cells for 48 hours and then a luciferase assay was performed. **p < 0.01 vs. Ctrl, ^##p < 0.01 vs. WT. The data are expressed as the means ± SEM of three independent experiments. (b) Multiple species contain a potentially conservatively acetylated lysine residue. The Nkx2.5 protein sequence from multiple species was BLASTed based on the reversibly acetylated lysine located at amino acid 182 (*) in mice. (c) Western blot showing that mutation of lysine 182 reduces the acetylation level of Nkx2.5. Myc-tagged wild type Nkx2.5 (Myc-WT) or mutant Nkx2.5-K182R (Myc-K182R) was expressed in HEK293A cells for 48 hours and were transfected into HEK293A cells for 48 hours and then immunoprecipitation and western blot were performed with the indicated antibodies. (d) Tandem mass spectrometry (MS/MS) analysis of Nkx2.5 peptide (AA residues 176–195) demonstrates that the acetylated lysine 182 of Nkx2.5 is deacetylated by SIRT1.