Western blotting was performed as described previously [39 (link)–41 (link)]. The hippocampus was collected from deeply anesthetized rats (n = 8 per group, 40 mg/kg, i.p.) and snap-frozen in liquid nitrogen. Tissues were homogenized on ice in RIPA buffer containing protease inhibitor cocktail and phosphatase inhibitor cocktail (Sigma–Aldrich). After centrifugation, the supernatants were collected and denatured with SDS–PAGE loading buffer for 5 min at 95°C. Equal amounts of protein were separated by SDS–PAGE and transferred to PVDF membranes (GE Healthcare Life Science). After blocking with 5% nonfat milk for 1 h at room temperature, membranes were incubated with primary antibodies overnight at 4°C (rabbit anti-pCREB, 1 : 1000, rabbit anti-CREB, 1 : 1000, Cell Signaling Technology; rabbit anti-BDNF, 1 : 600, Abcam). Then, the membranes were washed with TBST and incubated with secondary antibodies (1 : 1000, Santa Cruz Biotechnology) for 1 h at room temperature. Finally, bands were detected with an enhanced chemiluminescence reagent eECL Kit (CWBio, China) and quantified using ImageJ software (NIH, USA). GAPDH (rabbit antibody, 1 : 5000, Abcam) was used as the loading control.
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