Fluorometric assay kit

The deacetylase activity of SIRT1 was measured using a fluorometric assay kit (Enzo Life Sciences, Farmingdale, NY; Cat #: BML-AK500–0001). Cell extracts (75 μg total protein) were incubated with 5 μg of anti-SIRT1 antibody using the immunoprecipitation method described above. After the final wash, the samples were resuspended in 100 μl of assay buffer provided by the assay kit. The manufacturer’s instructions were followed and optimized for our samples. Briefly, the deacetylase reaction was initiated by adding 50 μM of Fluor de lys^® substrate and 1 mM nicotinamide adenine dinucleotide (NAD⁺; SIRT1 substrate) to samples. After incubating the samples for 20 minutes at room temperature, the reactions were stopped by addition of 1X Fluor de lys^® developer that contained either 1 μM trichostatin A (TricA; HDAC inhibitor) or 2 mM of nicotinamide (NAM ; SIRT1 inhibitor). Samples were read in a fluorometer (SpectraMax M5) at an excitation wavelength of 360 nm and emission wavelength of 460 nm.