Mouse anti β actin

NRSF was measured after SOD and MDA assays. Total proteins in the collected hippocampal tissues were isolated using cell lysis solution (Beyotime). Protein concentrations were determined by Bicinchoninic Acid assays (Beyotime). Equal amounts of protein were separated on 10% sodium dodecyl sulfonate-polyacrylamide gels and transferred to polyvinylidene fluoride membranes. The membranes were blocked in Tris buffered saline containing nonfat milk (10%) and 0.1% Tween-20 for 1 hour. The following primary antibodies were used: polyclonal rabbit anti-NRSF (Bioss) diluted 1:500, and mouse anti-β-actin (Beyotime) diluted 1:1000. After incubation with primary antibodies for 24 hours at 4°, the membranes were washed three times with Tris buffered saline containing 0.1% Tween-20 for 5 minutes and then incubated with goat anti-rabbit and goat anti-mouse IgG horseradish peroxidase antibody (Beyotime) at a dilution of 1:1000 for 1 hour at room temperature. The chemiluminescence signals were captured using a LAS 4000 mini (General Electric Company, Japan), and the bands were quantified by densitometry using the ImageJ system (National Institutes of Health, Maryland, USA). Grayscale values were measured and normalized to β-actin in the same sample.

Cells were harvested at indicated time and lysed by using RIPA lysis buffer on ice. A total of 20 μg protein was loaded on 7%−15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins were transferred to polyvinylidene difluoride membranes and incubated with the primary antibodies rabbit anti-USP7 (#4833, Cell Signaling Technology, CST, 1:1000), rabbit anti-p53 (#2527, CST, 1:1000), mouse anti-β-actin (#AA128, Beyotime Biotech., 1:3000) at 4 °C overnight. Then membranes were washed and incubated with corresponding secondary antibodies (Beyotime). Finally, the protein was visualized using an enhanced chemiluminescence detection kit (Beyotime). The density of the bands was analyzed, and the relative ratios of target genes and references were calculated.

Chemicals and antibodies were purchased as follows: resveratrol (Sigma, R5010) dissolved in 100% dimethyl sulfoxide (DMSO; Sigma, D2650) to a concentration of 40 mM, nicotinamide (Sigma, 72340) dissolved in phosphate-buffered saline (PBS) to a concentration of 200 mM, bafilomycin A (Sigma, B1793) dissolved in DMSO to a concentration of 5 μM, 0.25% trypsin solution (Sigma, 59429C), 0.2% type II collagenase (Sigma, V900892), Dulbecco's modified Eagle's medium and Ham's F-12 medium (DMEM/F12, 1:1; Gibco, 11320-033), fetal bovine serum (FBS; Gibco, 10099-141), penicillin-streptomycin (Sigma, G4664), rabbit anti-SIRT1 (Epitomics, ab32441), rabbit anti-Caspase3 (Epitomics, ab32351), rabbit anti-Collagen II (Abcam, ab34712), rabbit anti-LC3 (Cell Signaling Technology, 4599), mouse anti-Beclin-1 (Abcam, ab114071), mouse anti-β-actin (Beyotime, AA128), goat anti-rabbit IgG-HRP (Beyotime, A0208), goat anti-mouse IgG-HRP (Beyotime, A0216).