Figure 2.

Under conditions of excess nutrition, Sirt3 decline precedes down-regulation of Sirt6. A) H9c2 cardiomyocytes were treated with Pal (200 µM) for different time durations. Sirt3 and Sirt6 expression was analyzed by Western blotting. B) Quantitation of the Western blots. Note: Sirt3 down-regulation precedes down-regulation of Sirt6. Means ± se; n = 4. C) Sirt3 was immunoprecipitated after 2-h treatment of H9c2 cells with Pal, and protein acetylation was analyzed by Western blotting using anti–Ac-lysine antibody. D) The same protein lysate was subjected to Western blotting using specific antibodies to determine acetylation of MnSOD and OSCP. E–G) Western blots showing down-regulation of Sirt6 (E) and Sirt6 (F) in the hearts of db/db mice and mice fed an HF-HS diet (G) for 24 wk. H) Sirt3 was immunoprecipitated from hearts of mice fed a normal diet (ND) or HF-HS diet, and protein acetylation was analyzed by Western blotting using anti–Ac-K antibody. I) H9c2 cells were overexpressed with plasmids expressing mutant Sirt3 having K223 mutated to Q or to R. The acetylation of the Sirt3 target protein OSCP was analyzed by use of a specific anti–Ac-OSCP antibody. Ac, acetylated; Cont., control; IP, immunoprecipitation; WB, Western blot.