(A) Liver extracts from fed or fasted (24 hr) WT and SIRT3KO mice were immunoprecipitated with an HMGCS2 antiserum and analyzed by western blotting with antisera specific for acetyllysine; integrated density values were calculated and are shown relative to WT mice; data are represented in arbitrary units (AU) ± SEM, n = 3/condition; *p < 0.05.
(B) Recombinant HMGCS2 expressed in E. coli was incubated in vitro with recombinant SIRT3 or catalytically inactive SIRT3-H248Y in the presence or absence of nicotinamide, and the HMGCS2 acetylation status was assessed.
(C) Expression vectors for WT SIRT3, catalytically inactive SIRT3-H248Y, SIRT4, or SIRT5 were cotransfected into HEK293 cells with expression vectors for FLAG-tagged HMGCS2, and the levels of HMGCS2 acetylation were assessed.