Western bolt
Hippocampal tissues were prepared as previously described(W. Zhang et al., 2020) and homogenized with radioimmunoprecipitation assay lysis buffer containing protease and phosphatase inhibitors. The commercially available BCA protein assay kit (Boster, Wuhan, China) was used to determine protein concentrations. Then, protein samples were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). Bands were blocked with 5% BSA for 1.5 hours at room temperature. Primary antibodies were incubated overnight at 4 °C, and then, bands were washed with TBST and incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 2 hours. The antibodies used in this research include rabbit anti-BDNF (1:1000; Abclonal), anti-TrkB (1:1000; Abclonal), anti-p-TrkB (1:500; Abclonal), anti-Bax (1:1000; Abclonal), anti-Bcl-2 (1:1000; Abclonal), anti-ERK1/2 (1:1000; Cell Signaling Technology), anti-p-ERK1/2 (1:1000; Cell Signaling Technology), anti-CaMK2 (1:1000; Proteintech), anti-p-CaMK2 (1:500; Abclonal), anti-CREB (1:1000; Abclonal), anti-p-CREB (1:1000; Cell Signaling Technology), anti-GluR1 (1:1000; Abclonal), anti-p-GluR1 (1:500; Abclonal), anti-GAPDH (1:1000; Abclonal), and goat anti-rabbit (1:5000; Promoter). The bands were visualized using chemiluminescence (Pierce ECL Western Blotting Substrate, Abbkine) and measured by a computerized image analysis system (ChemiDoc XRS+, BIO-RAD, CA, USA).