SIRT1 deacetylase activity was evaluated in crude nuclear extract from heart samples [5 (link)]. Trichostain A (0.2 mM; Sigma-Aldrich, St. Louis, MO, USA), components of Fluor de Lys SIRT1 Fluorescent Activity Assay/Drug Discovery Kit (Enzo Life Sciences, Farmingdale, NY, USA), including 100 μmol/L fluorogenic peptide encompassing residues 379 to 382 of p53 with lysine 382 being acetylated, and 170 μmol/L NAD+ at 37°C for 1 h, followed by incubation in developer for 15 min at room temperature according to the manufacturer's instructions. Fluorescent intensity was measured using a Fluoroskan Ascent® microplate fluorometer (Thermo Electron Corp., Milford, MA, USA). No-enzyme and time 0 negative controls were generated by incubating developer II solution with 2 mmol/L NAM before mixing the substrates with or without samples. SIRT1 activity was calculated with the corrected arbitrary fluorescence units of the tested samples to noenzyme control and expressed as fluorescent units relative to the control. The nuclear-cytoplasmic fraction of heart tissue was conducted using the NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Thermo, Fisher Scientific, Rockford, IL, USA) [26 (link)].
Free full text: Click here
