Western blotting

Samples from PFC (n = 6/group) were dissociated in lysis buffer (containing protease inhibitors, 50 mM Tris–HCl, pH 7.6) on ice for 30 min and homogenized via ultrasonification (Ningbo scientz biotechnology CO. LTD, Ningbo, China). After centrifugation at 12,000 g for 10 min at 4 °C, supernatants were pooled and protein concentrations were measured with a BCA assay kit (Beyotime Institute of Biotechnology, China). Thirty μg protein samples were mixed with gel loading buffer (50 mM Tris-HCl, 10% SDS, 10% glycerol, 10% 2-mercaptoethanol, 2 mg/ml bromophenol blue) in a 1:1 ratio, boiled for 5 min and subjected to SDS-PAGE gels. The separated proteins were transferred to polyvinylidene fluoride membranes and incubated with mouse anti-Synaptophysin (1:1000, Cat.No.ab8049, Abcam, Cambridge, UK), rabbit anti-NR2B (1:1000, Cat.No.06-600, Millipore, USA), rabbit anti-NR2A (1:1000, Cat.No.4205, Cell Signaling Technology, USA), mouse anti-PSD-95 (1:1000, Cat.No.MAB1596, Millipore, USA), rabbit anti-GAPDH (1:1000, Cat.No.5174, Cell Signaling Technology, USA) and rabbit anti-β-tubulin (1:1000, Cat.No.2128, Cell Signaling Technology, USA) overnight at 4 °C. Signal was detected using HRP-conjugated rabbit or mouse antibodies followed by chemiluminescence using a Super Signal West Pico kit (Pierce) and Hyper film ECL (Amersham Biosciences). All experiments were carried out at least in triplicate. Autoradiography films were scanned and signals were quantified using Image J (NIH, USA). Density of each band was normalized to reference controls (β-tubulin or GAPDH).