Hippocampus tissues and hippocampal neurons were treated with ice-cold RIPA lysis buffer (Beyotime, Shanghai, China) to extract protein. After determining the protein concentrations using BCA Assay Kit (Beyotime), protein samples were separated in SDS-PAGE gels and then transferred onto PVDF membranes (Invitrogen). Subsequently, the membrane was incubated with skim milk, primary antibody against BDNF (1:2000, Boster, Beijing, China) or GAPDH (1:2000, Boster), and secondary antibody (Goat anti-rabbit, 1:10,000, Boster) in turn. The signal intensity was determined by Super ECL Detection Reagent (Yeasen, Shanghai, China).

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