Sirt1 Activity and Expression Evaluation

Sirt1 activity was evaluated from cytoplasmic compartment using a fluorometric assay (SIRT1 fluorimetric kit, BML-AK-555, Enzo Life Sciences, Villeurbanne, France) at the 15 minutes point. Expression of Sirt1 protein was assessed using western blot as previously described [18 (link)]. Briefly, 10 microgram of cellular extracts were resolved on 10% SDS-PAGE using a Mini-PROTEAN 3 Cell (Bio-Rad Laboratories, Hercules, CA, USA). The proteins were electrotransferred onto a polyvinylidene difluoride membrane (Amersham Biosciences, Saclay, France) using Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad Laboratories). The membranes were probed with primary antibodies followed by horseradish peroxidase-conjugated secondary immunoglobulin raised against the appropriate species; bands were detected using the ECL Plus kit (Amersham Biosciences). The primary antibodies used for western blot are as follows: rabbit anti-Sirt1 antibody (1:1000 dilution; Cell Signaling Technology, Beverly, MA) and mouse anti-beta-actin antibody (1:5000 dilution; Sigma-Aldrich, Saint-Louis, MO). Horseradish peroxidase-conjugated secondary antibodies goat anti-rabbit (1:5000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit anti-mouse (1:5000 dilution; DakoCytomation, Trappes, France) were used.