Western blot
A radioimmunoprecipitation (RIPA) assay buffer containing a mixture of protease and phosphatase inhibitors was used to extract the protein from the hippocampus. The protein concentration was measured by BCA Protein Assay kit (Thermo Fisher Scientific, USA) after brain cells had been suspended, homogenized, sonicated, and centrifuged for 15 minutes at 14,000g and 4 °C. Afterwards, proteins were boiled for 10 minutes at 100 °C in a buffer containing glycerol, Eagle’s medium, and SDS. After ten micrograms of protein had been loaded on 12.5% SDS-PAGE (Yeasen Biotech, China), the separated proteins were transferred to nitrocellulose membrane (Yeasen Biotech, China). The membrane was blocked by 5% BSA in the TBST buffer (10 mmol/L of Tris, pH 7.5; 100 mmol/L of NaCl; and 0.1% Tween 20), and was incubated overnight at 4 °C with specific primary antibodies (1:1000 dilution in 5% nonfat milk in the TBST buffer) of synaptophysin (CST, USA), PSD95 (CST, USA), β-tubulin (Proteintech, USA), PKA (Abcam, USA), p-PKA (Abcam, USA), CREB (Proteintech, USA) , p-CREB (Proteintech, USA), BDNF (CST, USA). On the second day, primary antibody was detected with anti-rabbi secondary antibodies (1:5000; Invitrogen, USA) and the immunoreactive bands were visualized with an enhanced chemiluminescence kit (Beyotime Biotechnology, China). Then Image J software was used to quantify the band density. A minimum of three blots were performed for each condition.