SIRT1 Activity Assay

Cells were grown to confluence in 6 cm petri dishes, differentiated for 9 days (peak adipogenesis in the second AIM induction) and 21 days (endpoint of adipogenic induction) using the adipogenic protocol with and without NAM treatment and harvested in CellLytic lysis buffer (Sigma-Aldrich) with 0.01% protease inhibitor (Sigma-Aldrich) added. Cell lysate was sonicated and centrifuged; the supernatant was flash frozen and stored at -80°C until assay.

SIRT1 protein was extracted from cell supernatant using immunoprecipitation. Briefly, a 1:50 dilution of SIRT1 antibody (Abcam) was added to each sample of cell supernatant and incubated overnight at 4°C with agitation. The antibody-supernatant mixture was added to a 50% slurry of protein A agrose bead (Cell Signaling) and incubated for 3 hours at 4°C with agitation. SIRT1 protein activity was then measured by fluorometric assay at 350 nm (Abcam) according to the manufacturer’s protocol for quantification of SIRT1 activity.

Total SIRT1 protein was measured simultaneously by Simple Western size-based protein assay (WES, ProteinSimple, Santa Clara, CA) following manufacturer’s protocol. Results from WES were analyzed using ProteinSimple Compass software. SIRT1 activity in each cell set was normalized to the cell set’s total SIRT1 protein as measured by WES.