Figure 4

Nkx2.5 is a target of SIRT1-dependent deacetylation.

(a) GST-Nkx2.5 and p300 were overexpressed in HEK293A cells, and then GST-Nkx2.5 was purified and subjected to an in vitro deacetylation assay in the presence/absence of recombinant SIRT1 and NAD. (b) Myc-Nkx2.5 was co-transfected with SIRT1, mutant SIRT1H363Y or SIRT2 into HEK293A cells for 48 hours, and then immunoprecipitation and western blot were performed with the indicated antibodies. (c) Myc-Nkx2.5 was co-transfected with increasing amounts of SIRT1 plasmid into HEK293A cell for 48 hours, and then immunoprecipitation and western blot were performed with the indicated antibodies. (d) Myc-Nkx2.5 was co-transfected with SIRT1 into HEK293A cells and the cells were then treated without or with 5 mM nicotinamide (NAM) for 48 hours. Immunoprecipitation and western blot were performed with the indicated antibodies. (e) Myc-Nkx2.5 was co-transfected without or with SIRT1 siRNA into HEK293A cells for 48 hours, and then immunoprecipitation and western blot were performed with the indicated antibodies. (f) Cardiomyocytes were infected with adenovirus-mediated shSIRT1 (Ad-shSIRT1) or control shRNA (Ad-shCtrl) for 48 hours, and then immunoprecipitation and western blot were performed with the indicated antibodies.