Glur1

Cell protein extracts were prepared using RIPA lysis buffer containing 50 mM Tris, 5 mM EDTA, 150 mM NaCl, 0.1% SDS, 0.5% Deoxycholate, 1% NP40, protease inhibitor cocktail (Roche) and phosphatase inhibitor (Pierce). Tissue protein extracts were prepared from frozen samples using 1% SDS (Invitrogen) in 50mM pH 8.0 Tris buffer containing protease inhibitor cocktail (Roche) and phosphatase inhibitor (Pierce) with brief sonication on ice. Electrophoresis, transfer, blocking and antibody preparations were performed as previously described (19 (link)). Primary antibodies included: NPTX2 (Sigma-Aldrich- PRS4573) and β-actin (Sigma-Aldrich-A5441), GluR1 (Santa Cruz-sc-55509), GluR2 (Santa Cruz-sc-7601), p-CAMK1 (Santa Cruz-sc-373853), total CAMK1 (Abnova-PAB2769), GluR3(Cell Signaling-D47E3), PARP (Cell Signaling-9542), HA-tag (Cell Signaling-3724) and Flag-tag (Cell Signaling-8146), GluR4 (Millipore-AB1508), total AKT (Cell Signaling-2972), and p-AKT (R&D Systems-AF887). Secondary species-specific horseradish peroxidase-labeled antibodies (Jackson Immunoresearch) were applied, and Supersignal chemiluminescent kit (Pierce) was used to perform detection.

Hippocampal or primary neuronal culture lysates were prepared by homogenizing in RIPA buffer containing protease inhibitors. Following BCA assay (Fisher Scientific), we separated 20–60μg of lysate through 10% polyacrylamide gels and transferred protein to nitrocellulose membranes (Fisher Scientific). Membranes were blocked with 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBST). Membranes were incubated in primary antibody (in 0.3% BSA in TBST) at 4°C overnight using antibodies to detect KL (KL2–34, 1:200 (Maltare et al. 2014 (link))), β–tubulin (1:10,000, DSHB, E7), synaptophysin (1:10,000, Abcam, AB52636, Cambridge, MA), GABA(A) α1 receptor (1:5000, Alomone labs, aga–001, Jerusalem, Israel), GluN1 (1:500, Neuromab, 75–272, Davis, CA), GluN2b (1:1000, Millipore, 06–600, Billerica, MA), GluR1 (1:500, Santa Cruz, sc55509, Dallas, TX), GluR2 (1:500, Neuromab, 75–002), PSD–95 (1:500, Abcam, ab18258, Cambridge, MA), v-Glut (1:500, Neuromab, 73–006) and His Tag (1:1000, Fisher Scientific, MA1–21315). Secondary antibodies conjugated to HRP were detected by chemiluminescence (Immobilon, Millipore or Supersignal, Fisher Scientific) and exposure to film. Band intensity was quantified using Image J (Schneider et al. 2012 (link)).