Fig. 1. SIRT1 is an active nuclear NAD-dependent HDAC. (A) Equivalent amounts of wild-type His-SIRT1 or mutant His-SIRT1H363Y were tested for HDAC activity in the presence (full column) or absence (open column) of 1 mM NAD⁺. Histone deacetylase activity is given as radioactivity (c.p.m.) of [³H]acetate released from an acetylated histone H4 peptide. Control assays containing only acetylated H4 peptide without any recombinant protein were performed. (B) NAD-dependent HDAC assay performed essentially as above in which GST–SIRT1 was tested for activity in the absence (2) or presence of either 2 µM TSA (3) or 5 mM nicotinamide (4). All reactions contain identical amounts of GST–SIRT1. (C) Asynchronous HeLa cells were analyzed for endogenous SIRT1 expression using an anti-SIRT1 antibody followed by incubation with an Alexa 488-conjugated secondary antibody (green). Nuclear DNA was stained with propidium iodide (red). Note the high level of propidium iodide staining in the nucleoli.