Glur1

The PSD95 immunoprecipitation assay was probed with PSD-95 rabbit primary antibody (1:1000, Cell Signaling) to check for presence of PSD-95 pulled down. Equal amounts of sample were loaded to probe interaction of PSD-95 with GluR2 (Millipore), GluR1, and ILK with rabbit primary antibodies (1:1000, Cell Signaling). Whole hippocampal protein lysates were probed for BDNF or proBDNF, and GSK3β to total GSK3β, using their respective primary antibodies at 1:1000 (Cell Signaling). All blots were probed with Dy-Light 660 anti-rabbit secondary antibody (1:10000, Thermo Scientific) using a Fuji FLA 5100 scanner. They are presented as means ± SEM. Significance was determined using a two-tailed Student's t-test.

The whole hippocampus (ventral and dorsal) was lysed in RIPA buffer containing protease inhibitors and phosphatase inhibitors. Protein concentration was determined colorimetrically by NANODROP 2000 (Thermo Scientific). Protein lysates were separated by 10% SDS-PAGE electrophoresis and were transferred onto polyvinylidenedifluoride (PVDF) membranes. After blocking with 5% BSA for 1 hr, and the following antibodies were used: NR1(Santa Cruz Biotechnology, 5704S, 1 : 500), AKT/p-AKT (Cell Signaling Technology, 9272S, 4058s, 1:500), mTOR/p-mTOR (Cell Signaling Technology, 2972S, 2971S,1:500), p70S6 kinase p70s6k) / p--p70S6K (Cell Signaling Technology, 2708S, 9234S, 1:500), p-4EBP1 (Cell Signaling Technology, 9644S.1:1000), GluR1 (Cell Signaling Technology, 12551S, 1:1000) and β-tubulin (Epitomics, 1879-1, 1:2000). After the blots were incubated with antibodies overnight at 4 °C, they were incubated with horseradish peroxidase- conjugated secondary antibodies for 1 hr. The blots were visualized using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific Inc.). The resulting data was analyzed statistically using SPSS Statistics 20. All experiments were performed in troplicate. The final data are expressed as a ratio of the relative optical density (ROD) of the protein of interest to the ROD of β-tubulin. A p-value < 0.05 was considered significant.

The hippocampus of mice was dissected and sonicated in a lysis buffer. SDS—PAGE separated the extracts. The transferred membrane was blocked with 3% bull serum albumin and then incubated in primary antibodies (CRMP1 (GTX114940; GeneTex, Irvine, CA, USA), CRMP2 (ab129082; Abcam, Cambridge, UK), CRMP3 (ab36217; Abcam), CRMP4 (13661-1-AP; Proteintech, Rosemont, IL, USA), CRMP5 (GTX19352; GeneTex, Irvine, CA, USA), GluR1 (CST#13185; Cell Signaling Technology, Beverly, MA, USA), GluR2 (CST#13607; Cell Signaling Technology, Beverly, MA, USA), p-GR (CST4161; Cell Signaling Technology, Beverly, MA, USA), and NR3C1 (GR) (A2162; ABclonal, Woburn, MA, USA), and β-Actin (MAB1501; Millipore, Burlington, MA, USA) used as internal control) overnight at 4 °C. Next, after three times washing with TBST for 10 min, the membrane was incubated with secondary antibodies for 1 h at room temperature. Protein levels were detected and quantified by a BioImaging System (UVP Inc., Upland, CA, USA).