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. Author manuscript; available in PMC: 2012 Dec 20.
Published in final edited form as: Cell. 2008 Jul 25;134(2):329–340. doi: 10.1016/j.cell.2008.07.002

Figure 6. SIRT1 Regulates BMAL1 Lys537 Acetylation.

Figure 6

(A) Class III HDAC inhibitor enhances BMAL1 Lys537 acetylation. JEG3 cells transfected with Myc-CLOCK and Flag-Myc-BMAL1 were treated with HDAC I and II inhibitor, TSA (1 mM), for 6 hr and HDAC III inhibitor, NAM (10 mM), for 16 hr before harvest. Immunoprecipitated BMAL1 proteins by FLAG antibody were subjected to SDS-PAGE and probed with acetylated BMAL1 or Myc antibodies.

(B) SIRT1 deacetylates acetylated Lys537 in BMAL1. JEG3 cells transfected with Myc-CLOCK and Flag-Myc-BMAL1 were cotransfected with SIRT1-Flag, SIRT2-Flag, or SIRT3-Flag. Immunoprecipitated BMAL1 proteins by FLAG antibody were probed with acetylated BMAL1 or Myc antibodies. Immunoprecipitated SIRT proteins by FLAG antibody were probed with FLAG M2 antibody.

(C) Acetylated BMAL1 is not deacetylated by deacetylase-deficient mutant SIRT1. JEG3 cells transfected with Myc-CLOCK and Flag-Myc-BMAL1 were cotransfected with WT or mutant (H363Y) SIRT1-HA. Immunoprecipitated BMAL1 proteins by FLAG antibody were probed with acetylated BMAL1 or Myc antibodies. SIRT1 protein amount was detected by SIRT1 antibody.

(D) Deacetylation of BMAL1 by SIRT1 is NAD+ dependent manner. JEG3 cells transfected with Myc-CLOCK and Flag-Myc-BMAL1 and SIRT1-HA were treated with 1 mM NAD+ or 10 mM NAM for 16 hr before harvest. Immunoprecipitated BMAL1 proteins by FLAG antibody were probed with acetylated BMAL1 antibody. SIRT1 protein amount was detected by SIRT1 antibody.