A specific antibody that recognizes BMAL1 only when it is acetylated at K537. (A) We have raised a polyclonal antibody against an 11 amino acid peptide in which K537 is uniquely acetylated. The antibody has been successfully tested in western analysis, immunoprecipitations, and immunohistochemistry. (B) Results of a transfection in 293 cells in which CLOCK, BMAL1, or a BMAL1(K537R) mutant is ectopically expressed. Western analysis shows that the antibody specifically recognizes BMAL1 when it is acetylated and that CLOCK elicits this event (Hirayama et al. 2007). (C) SIRT1 deacetylates BMAL1(K537) in vitro. Acetylated BMAL1 was prepared from HDAC inhibitor-treated JEG3 cultured cells transfected with Flag–Myc–BMAL1 and Myc–CLOCK. Recombinant SIRT1 and deacetylation buffer were used from the SIRT1 Fluorimetric Activity Assay/Drug Discovery Kit (AK-555; BIOMOL International). Immunoprecipitated (IP) AcBMAL1 and recombinant SIRT1 were incubated in deacetylation buffer with 5 mM NAD+ or 10 mM nicotinamide (NAM) for 90 min at 37°C. SIRT1 is able to deacetylate BMAL1 only in the presence of NAD+.