Enhanced chemiluminescence ecl detection system

The expression level of Nrf2 was determined by western blot analysis. Cells were collected and cytosolic and nuclear proteins were extracted using a commercial nuclear and cytoplasmic protein extraction kit (Beyotime Biotechnology, Beijing, China) according to the manufacturer's instruction. The proteins were denatured by heating in the water bath at 95°C for 5 min, and separated on 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane (Millipore, USA). The membranes were blocked with 5% skim milk dissolved in PBST at room temperature for 1 h and incubated with anti-Nrf2 and anti-Lamin B1 mAbs (Abcam, Cambridge, MA) at 4°C overnight. The membranes were washed with PBST for three times and incubated with an HRP-conjugated secondary antibody (Sigma, USA) for another 1 h at room temperature. After washing with PBST three times again, positive reactions were detected by an enhanced chemiluminescence (ECL) detection system (Beyotime Biotechnology, Beijing, China). The band intensities were quantified using a VILBER Fusion FX5 Luminescence image analysis system (Vilber Lourmat, France) and compared with different groups.

The hippocampal tissue was placed in the RIPA lysate with PMSF protease inhibitor for homogenization, followed by centrifugation at 12,000 rpm at 4 ℃ to collect the supernatant. A Bicinchoninic acid (BCA) Protein Assay Kit (Beyotime, P0010, Shanghai, China) was used for measuring the protein concentration, and the proteins were subsequently treated with RIPA lysis buffer for further processing. The sample was then boiled for 10 min, cooled and subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) for separation. The protein that has been isolated was moved onto a polyvinylidene fluoride (PVDF) membrane from Merck Millipore (ISEQ00010, USA). After an hour of using 5% skim milk to block the PVDF membrane. And then it was exposed to primary antibodies: SIRT1 (1:1000, 9475S, Cell Signaling Technology), BDNF (1:1000, ab108319, Abcam), and GAPDH (1:1000, ET1601-4, HUABIO) overnight in cold storage at 4 ℃. The membranes were then treated with an antibody conjugate with horseradish peroxidase (1:2000, Beyotime) for an hour at room temperature. The protein bands were then subjected to a quantitative analysis utilizing ImageJ software and an Enhanced Chemiluminescence (ECL) detection system (Beyotime).