Protein Expression Analysis in Cell Signaling

According to the manufacturer's protocol [19 (link)], proteins were extracted using RIPA Lysis Buffer (Beyotime, China) and protein concentration was measured using a bicinchoninic acid assay (Beyotime, China). Subsequently, the prepared protein was separated by 12% polyacrylamide-SDS gels and then transferred onto PVDF membranes (Roche, Switzerland). After blocked with skim milk, the PVDF membranes were subjected to incubation with primary antibodies: Bcl-2, Bax, cleaved-Caspased-3, GCR, SIRT1, PGC-1α, and BDNF (1 : 500, Wanleibio Wuhan, China) overnight at 4°C. On the following day, the membranes were incubated with the secondary antibody at 37°C for 45 min and the intensity of protein expression was detected by ECL chemiluminescence (Beyotime, Beijing, China). Protein expression levels were semiquantified using ImageJ software (version 1.8.0; National Institutes of Health).