FIGURE 3.

SIRT3 deacetylates and activates CerS1, -2, and -6. CerS1, -2, and -6 were immunoprecipitated from baseline cerebral mitochondria of Sirt3 KO mice. CerS1 (A), CerS2 (B), and CerS6 (C) containing immunocomplexes (IC) were incubated with active recombinant SIRT3 in the presence or absence of NAD⁺ and NAM, a sirtuin inhibitor. In all panels, the same immunoprecipitation procedure was performed except for the primary antibody application as a control (IgG). After deacetylation, ICs were analyzed by Western blot using anti-CerS1 (Santa Cruz Biotechnology), anti-CerS2 (Santa Cruz Biotechnology), anti-CerS6 (Novus), anti-acetyl-lysine (Cell Signaling), and anti-SIRT3 (Cell Signaling) antibodies or by tandem MS (D) to determine the corresponding ceramide synthase activity as described under “Experimental Procedures.” Data are means ± S.E., *, p < 0.05, n = 4.