Glun2a

Four sets of synaptosome fraction samples from 3-month-old Aβ-GFP Tg and non-Tg mice were prepared as described previously^{53 (link)}. Each set was a mixture of 3 individual Aβ-GFP Tg or non-Tg littermate hippocampi (totally 12 mice were used both in Aβ-GFP Tg or non-Tg mice). Proteins were subjected to SDS-PAGE and transferred to PVDF membranes. VAMP-2 (1:5000, Abcam, Cambridge, UK), Munc13-1(1:1000, Synaptic Systems, Gottingen, Germany), syntaxin-1 (1:1000, Merck), synaptophysin (1:2000, Sigma-Aldrich), PSD-95 (1:200, Merck), GluN1 (1:200, Merck), GluN2A (1:500, Merck), GluN2B (1:200, Merck), GluA2 (1:200, Merck), neuroligin 1(1:500, Synaptic Systems), and actin (1:2000, Merck) antibodies were used to probe the corresponding proteins and ECL was used for detection (Immunostar Zeta and Immunostar LD, FUJIFILM Wako Pure Chemical Corporation).
Immunoblotting of homogenates of individual Aβ-GFP Tg and non-Tg mouse hippocampi was performed using 6E10, GFP (1:200), GluN2B (n = 5 each for statistical analyses), tau (n = 8 each for statistical analyses, phospho T231, 1:1000, Abcam), and actin antibodies.
Immunoblot results were quantified via a C-Digit blot scanner (LI-COR Biosciences, NE, USA) and normalized with actin or GAPDH (supplementary Fig. S7) and the expression ratios of Aβ-GFP Tg were calculated with the values of non-Tg mice as 1.

Samples were resolved on 8% SDS-PAGE, transferred onto nitrocellulose membranes (Bio-Rad), and incubated with primary antibodies against STEP (1:1000, Millipore), non-phospho-STEP (1:1000, Cell Signaling), phospho-Tyr (1:2000, Millipore), GluN2A (1:2000, Millipore), GluN2B (1:2000, Millipore), pY416 Src (1:1000, Cell Signaling), Src (1:2000 Santa Cruz), Fyn (1:2000 Santa Cruz), and β-actin (1:5000, Santa Cruz) overnight at 4 °C, and HRP-conjugated secondary antibodies (1:5000; Pierce) or Clean-Blot IP detection reagent HRP (1:1000; Thermo Scientific, Rockford, IL) for 2 h at RT. Immunoreactivity was developed with a Chemiluminescent Substrate kit (Pierce) and visualized by G:BOX with the GeneSnap software (Syngene, Cambridge, UK). All densitometric quantification was obtained using ImageJ software (National Institutes of Health).

Four mice per group were anesthetized with intraperitoneal injections of zoletil (90 mg/kg) and xylazine (10 mg/kg), and their hippocampi were collected. The extracted proteins were quantified by the BCA Protein Assay System (Pierce, USA). The proteins were subjected to SDS-PAGE, followed by electro-transfer to PVDF membranes (Millipore, USA). After blocking with 5% skim milk in 0.1% Tris-buffered saline (TBS)/Tween-20, the blots were probed with primary antibodies against GluA1 (1:1000, Abcam), GluN1 (1:1000, Sigma-Aldrich), GluN2A (1:2000, Millipore), NR2B (1:500, BD Transduction Laboratories), and synaptophysin (1:4000, Abcam) overnight at 4 °C. After incubation, the membranes were subsequently incubated for 2 h at room temperature with the appropriate secondary antibodies conjugated with horseradish peroxidase (Vector, USA). The proteins were visualized with an enhanced chemiluminescence kit (Pierce, USA) and band intensities were quantified using the Kodak Gel Logic 2200 imaging system with Molecular Image analysis software (Kodak, USA). β-Actin was used as a loading control (1:10000, Sigma-Aldrich). Data are presented as an average of at least three experiments and analyzed for statistical significance by using Mann-Whitney tests (Prism; Graph Pad, USA).

For the detection of glutamatergic receptors, cells grown in microfluidic chips were homogenized in lysis buffer (0.5% Triton, 0.5% sodium dodecyl sulfate, 50 mM Tris HCL 150 mM NaCl) and protease inhibitor mixture (Invitrogen) on ice. Samples were analyzed with SDS-PAGE, followed by western blotting. For the detection of dopamine receptors, cells grown in 24-well plates were lysed directly in SDS buffer, heated to 95 °C for 3 min, and after cooling urea was added to samples until a final concentration of ~4 M. Samples were run on ultrathin (0.1 mm) gels^{50 (link)}, and Western blots revealed with alkaline phosphatase-coupled secondary antibodies and NBT/BCIP reagent. The following antibodies were used for western blotting: GluN1 (05–432, Millipore, 1/1000), GluN2A (Millipore, 04–901, 1/500), GluN2B (Millipore, 05–920, 1/500), VGLUT1 (gift from Dr Salah El Mestikawy, IBPS CNRS UMR8246, Paris, France, 1/1000), D1R (Sigma, D2944-100, 1/500) and D2R (Millipore, ABN462, 1/500).

In addition to the NRG2 antibodies described above, commercial and custom antibodies against the following proteins were used in this study: ErbB4, rabbit monoclonal mAB10 (1 μg ml⁻¹; ref. 7 (link)) and rabbit polyclonal custom antibody 5721 (unpublished; 0.4 μg ml⁻¹); Kv2.1, mouse monoclonal (clone K89/34; NeuroMab; 1 μg ml⁻¹); rabbit polyclonal antibody against NRG1 (SC-348; Santa Cruz Biotechnology; 0.2 μg ml⁻¹); anti-V5 epitope tag (AbD Serotech; 1 μg ml⁻¹); Bassoon, rabbit polyclonal (Synaptic Systems; 1:1,000); Gephyrin, mouse monoclonal (clone mAb7a; Synaptic Systems; 1:500); PSD95, mouse monoclonal (clone 7E3-1B8; Pierce; 1:500); Calbindin, mouse monoclonal (clone CB-955; Sigma; 1:1,000); GFP, rabbit polyclonal (Molecular Probes; 1:2,000); Erk2, rabbit polyclonal (C-14; Santa Cruz; 0.2 μg ml⁻¹); phospho-Erk2, mouse monoclonal (clone E-4; Santa Cruz; 0.2 μg ml⁻¹); GAPDH, mouse monoclonal (clone 6C5; Santa Cruz; 0.2 μg ml⁻¹); clathrin heavy chain, mouse monoclonal (SC-12,734; Santa Cruz; 0.1 μg ml⁻¹); GluN2A, rabbit monoclonal (clone A12W; Millipore; 1:2,000); GluN2B (clone 13; BD Biosciences; 1 μg ml⁻¹); and GluA1 (AB1504; Millipore; 0.2 μg ml⁻¹).