Mab363

The cell-based assay consisted of HEK293 cells transfected with plasmids expressing GluN1/N2B in equimolar ratios as described previously (14 (link)). Cells were grown for 24 hours after transfection and in the presence of ketamine (500 μM) to prevent cell death after transfection. Transfected cells were then fixed with 4% paraformaldehyde (5 minutes at room temperature), permeabilized with 0.3% Triton X-100 (5 minutes at room temperature), incubated with serum diluted 1:40 (2 hours at room temperature), then washed with phosphate-buffered saline and incubated with a mouse monoclonal antibody against a noncompeting GluN1 epitope located in the extracellular loop at amino acid 660–811 (dilution 1:20,000; MAB363, Billerica, Mass., Millipore) for 1 hour at room temperature, followed by the corresponding fluorescent secondary antibodies (Alexa Fluor 488 goat antihuman IgG, Invitrogen A11013, diluted 1:1,000; Alexa Fluor 594 goat antimouse IgG, Invitrogen A11032, diluted 1:1,000) for 1 hour at room temperature. Cell-based assays for AMPA receptor, GABAA receptor, GABAB receptor, mGluR5, LGI1, Caspr2, DPPX, D₂ receptor, and glycine receptor were performed as previously described.

For IP validation experiments, primary neurons were lysed and IP was conducted as described in (Guo et al. 2015 (link)). Briefly, 200μg of protein was incubated with IP antibody (Homer1 AT1F3 from LS Bio or mGluR 5675 from Millipore) overnight at 4°C, then with protein G magnetic beads (New England Biolabs) for 1 hour. After washing three times in PBS, samples were heated in 3X Laemmli loading buffer and subjected to SDS-PAGE electrophoresis followed by Western blotting. For all Western blot experiments, gels were transferred onto PVDF (Millipore) membranes, blocked in 4% milk or BSA in TBST (0.05M Tris, 0.15 M NaCl, pH7.2, 0.1% (v/v) Tween20) for 60 minutes at room temperature, and primary antibodies were applied overnight at 4°C in blocking medium. Primary antibodies for Western blots were diluted as follows: mGluR5 (ab5675, Millipore, 1:1000), NMDAR1 (MAB363, Millipore, 1:1000), Homer1 (D-3, Santa Cruz, 1:500), PSD-95 (K28/43, NeuroMab, 1:1000) phosphoERK1/2 (197G2, Cell Signaling, 1:2000), and ERK1/2 (137F5, Cell Signaling, 1:2000). After washing and probing with the appropriate species-specific secondary horse radish peroxidase-conjugated antibodies, blots were imaged using Pierce Femto detection reagents in a Protein Simple western blot imaging system.