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).