SIRT1 deacetylase activity detection

SIRT1 deacetylase activity of the isolated injured-side cortical tissue was performed as previously described using the Cyclex SIRT1/Sir2 Deacetylase Fluorometric Assay Kit (Catalog number CY-1151; Cyclex, Nagano, Japan) (Li et al., 2015b). Briefly, the cortex tissue (100 mg) was homogenized in 500 μL of immunoprecipitation buffer (T-PER Tissue Protein Extraction Reagent 78510; Pierce, Rockford, IL, USA). For immunoprecipitation assay of SIRT1, primary antibody against SIRT1 (10 μg) was incubated overnight with precleared lysates (800 μg) at 4°C before the addition of 20 μL of protein agarose A/G beads (Protein A/G PLUS agarose: sc-2003; Boston, MA, USA). The reactions were rotated for 2 hours at 4°C. The beads were extensively washed and centrifuged followed by a deacetylation assay. The final reaction mixture (50 μL) contained 50 mM Tris-HCl (pH 8.8), 0.5 mM dithiothreitol, 0.25 mAU/mL lysyl endopeptidase, 1 μM trichostatin A, 200 μM NAD⁺ and 10 μL of tissue extraction. The mixtures were mixed well and incubated for 30 minutes at room temperature. The fluorescence intensity (excitation wavelength 340 nm, emission wavelength 460 nm) was measured using an automatic microplate reader (SpectraMax M5, Shanghai, China).