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.