Cellular SIRT1 activity assay

The celluar SIRT1 activity in an immunoprecipitate was performed using SIRT1 Activity Assay Kit Fluorometric (ab156065) according to the manufacturer’s instructions. Treat cells by adding fresh media containing test compound for desired time. To harvest cells under non-denaturing conditions, remove media and rinse cells once with ice-cold phosphate-buffered saline (PBS). Remove PBS and add 0.5 mL 1Xice-cold Cell Lysis Buffer to each plate (10 cm dish) and incubate the plate on ice for 5 min. Scrape cells off the plate and transfer to microcentrifuge tubes. Sonicate four times for 5 s each on ice. Microcentrifuge for 10 min at 4 °C, and transfer the supernatant to a new tube to obtain the cell lysate. Take 200 μL cell lysate and incubate with a suitable anti-SIRT1 antibody or anti-IgG according to the manufacturer’s instructions. Add Protein A agarose beads (20 μL of 50% bead slurry). Incubate with gentle rocking for 1–3 h at 4 °C. Microcentrifuge for 30 s at 4 °C. Wash pellet three times with 500 μL of 1X Cell Lysis Buffer and once with 500 μL of SIRT1 assay buffer (50 mM Tris-HCl (pH 8.8), 0.5 mM dithiothreitol). After immunoprecipitation, add reaction mixture containing Fluoro-Substrate peptide solution to Protein A agarose beads as an “Enzyme Sample” and measure NAD-dependent deacetylase activity.