Anti nr1

Ipsilateral hippocampal tissues from sham and CCI injured mice were homogenized in RIPA buffer with protease and phosphatase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO) and benzonase nuclease (Millipore Corporation, Billerica, MA) and mixed by rocking at 4 °C for at least 15 min. Tissues were centrifuged, supernatant was recovered, samples were diluted and standardized to protein concentrations. Protein samples were separated on 8–10% SDS-PAGE gel and transferred to a nitrocellulose membrane. Membranes were then blocked with 5% milk or 5% BSA in 0.1 M phosphate buffer with 0.1% Tween-20 for 1 h at room temperature (RT) and incubated overnight at 4 °C with primary antibodies. Membranes were incubated for 1 h at RT with HRP-conjugated secondary antibodies (Jackson Immunoresearch Laboratories, West Grove, PA). Bands were visualized using SuperSignal substrate (ThermoScientific, Pittsburg, PA). The following primary antibodies were used: anti-GluR1, anti-NR1, anti-NR2B (EMD Millipore, Billerica, MA), anti-GFAP (BD Biosciences, San Jose, CA), anti-SNAP25, anti-SNAP23 (ABCAM, Cambridge, MA) and anti-β-tubulin (Sigma-Aldrich, St. Louis, MO) antibodies. ImageJ was used to perform density analysis. Protein measurements were standardized to β-tubulin and normalized to average WT sham signals.

Surface biotinylation was performed according to the previous studies (Kim et al., 2015a (link); Kim et al., 2015b (link); Kim and Ziff, 2014 (link); Sztukowski et al., 2018 (link)). Equal amounts of protein were loaded on 10% SDS-PAGE gel and transferred to nitrocellulose membranes. Membranes were blotted with anti-NR1 (Millipore, 1:1000), anti-GluA1 (Millipore, 1:2000), anti-GluA2 (Abcam, Cambridge, UK, 1:2000), anti-phosphorylated GluA1 S845 (Millipore, 1:1000) and anti-actin (Abcam, 1:2000) antibodies and developed with Enhanced Chemiluminescence (ECL) (Thermo Fisher Scientific, Waltham, MA). Immunoblots were at least duplicated for quantitative analysis.