The levels of SIRT1 do not cycle, but its deacetylase activity does. (A) To assess whether SIRT1 protein levels oscillate in a circadian manner, nuclear extracts from mouse liver were prepared following various methods. Western blot (WB) analyses using specific antibodies demonstrated that SIRT1 levels displayed marginal or no oscillation. Nuclear extracts (NE) were prepared following, using a nuclear lysis buffer containing 450 mM NaCl (high salt) or 150 mM NaCl (low salt), following the method by Andrews and Faller (1991); alternatively, nuclear extracts were prepared using the method of Gorski et al. (1986). Nuclei were purified over a sucrose cushion and the resulting nuclear pellet was extracted. Nuclei were lysed with ammonium sulfate and chromatin was centrifuged out. Nuclear proteins were precipitated by addition of 0.3 g/mL solid ammonium sulfate. Nuclear extracts were also prepared using the commercially available kit NE-PER Nuclear and Cytoplasmic Extraction Reagents (PIERCE Biotechnology). In all of these conditions, the SIRT1 protein did not show oscillation (see also Nakahata et al. 2008). (B) Mouse liver total extracts were used to immunoprecipitate SIRT1. SIRT1 levels were noncyclic in total extract at different times of the circadian cycle (lower panel). The same immunoprecipitates were subjected to deacetylase enzymatic assays as described (Nakahata et al. 2009). The histone deacetylase (HDAC) activity of SIRT1 followed a circadian profile.