Anti glua2

Hippocampi of mice were homogenized in 25 mM HEPES + Protease-Inhibitor Cocktail (Roche). Lysates were centrifuged for 5 min at 2000 rpm. Protein concentrations in supernatants were determined in triplets with BCA-Kit (Pierce). From each lysate 8 μg protein was separated by 7% SDS-PAGE and proteins were transferred to nitrocellulose membranes. The blotted proteins were probed with polyclonal antibodies against GluA1 (Merck Millipore 0.1 μg/ml), anti-GluA2 (Merck Millipore 0.16 μg/ml), anti-GluN1 (Merck Millipore, 0.25 μg/ml, monoclonal anti-GluA3 (ThermoFisher, 1:250), anti-CaMKII (MAB 8699 Merck Millipore 0.1 μg/ml) and anti-ß-actin (AC-15 Ascites, Sigma, 1:25,000), followed by peroxidase-linked anti-rabbit or -mouse secondary antibodies (Jackson Immuno Res., 1:20,000). ECL+ (ThermoFisher) was used to visualize immuno-labeled proteins.

After the behavioral experiment, the mouse brain was quickly removed, and hippocampal tissues (six per group) were placed in ice-cold saline. The tissues were homogenized with RIPA buffer and protease inhibitors for 10 min and then centrifuged at 12,000 rpm for 30 min at 4 °C. The supernatants of the samples were assayed for protein content, diluted with 1:4 sample buffer and heated at 95 °C for 5 min. The proteins were loaded onto 10% sodium dodecyl sulfate (SDS) polyacrylamide gels and then transferred onto PVDF membranes. The membranes were incubated with blocking buffer (5% nonfat milk and 0.1% Tween-20 in Tris-buffered saline [TBST]) for 1 h at 27 °C. The membranes were incubated with the following primary antibodies overnight at 4 °C: anti-GluA1 and anti-GluA3 (Abcam 1:2000), anti-GluA2 (Millipore 1:2000), anti-NR1 (Sigma 1:1000), anti-NR2A and anti-NR2B (Abcam 1:2000), anti-ERK (Abcam 1:1000), anti-CREB (Abcam 1:1000), and anti-PSD95 (CST 1:1000). After three washes with TBST, the membranes were incubated with IRDye 700DW- or 800DW-conjugated anti-mouse or anti-rabbit IgG (1:10,000) for 1 h at room temperature, washed with PBS, and scanned to detect fluorescence with the LI-COR Odyssey detection system.
The investigators were blinded to the group assignment of the animals in all of the aforementioned experiments.

The antibodies used were as follows: anti-cortactin (clone 4F11, Millipore); anti-GluA1 (Calbiochem); anti-GluA2 for Westerns (Synaptic systems); anti-GluA2 for co-IPs (MAB397, Millipore); anti-GluA2 for immunocytochemistry (BD Pharminogen, mouse); anti-GluA3 (Alomone, rabbit); anti-phosphoY421-cortactin (Sigma); anti-phosphoY466-cortactin (Millipore); anti-phosphoY482-cortactin (Millipore); anti-Myc (Santa Cruz); anti-Flag (Sigma); anti-GST (Cell Signalling Technologies); anti-GFP (Chromtek); anti-LAMP1 (Abcam); anti-PSD-95 (Millipore); control IgG (Thermo).

Whole and synaptosomal lysates of the mouse brains were prepared as described previously (Han et al., 2009 (link); Jin et al., 2019a (link),b (link)). The antibodies used for Western blot analysis were anti-Cofilin (1:1,000, Abcam, AB42824), anti-CYFIP1 (1:1,000, Millipore, AB6046), anti-CYFIP2 (1:3,000, Abcam, AB95969), anti-GABA-A-R-β2/3 (1:1,000, NeuroMab, 75-363), anti-GAPDH (1:3,000, Cell Signaling, #2118), anti-Gephyrin (1:500, Synaptic Systems, 147-011), anti-GluA1 (1:2,000, Millipore, 04-855), anti-GluA2 (1:1,500, Millipore, MAB397), anti-GluN1 (1:1,000, Millipore, MAB363), anti-Homer1b/c (1:1,000, Synaptic Systems, 160-002), anti-Neuroligin-3 (1:1,000, NeuroMab, 75-158), anti-PSD-95 (1:2,000, Thermo Fisher Scientific, MA1-046), and anti-WAVE1 (1:1,000, NeuroMab, 75-048). Western blot images were acquired using a ChemiDoc Touch Imaging System (Bio-Rad) and quantified using ImageJ software.

Anti-GluK3 antibody raised in rabbit against C-terminal 17 amino acid residues of mouse GluK3 (903–919, NM_001081097) was used for immunohistochemistry, immunoprecipitation, and western blotting. The rabbit polyclonal antibodies anti-GluK2 (Synaptic Systems, 180 003), anti-GluK4^{41 (link)}, and anti-GluK5 (Millipore, 06-315) were used for immunoprecipitation and western blotting. The specificities of these antibodies have been determined previously^{29 (link)}. Anti-GluN1 (BD Biosciences, 556308), anti-GluN2A (BD Biosciences, 612286), anti-GluN2B (BD Biosciences, 610416), anti-GluA1 (Frontier Institute, Japan, MSFR102270), anti-GluA2 (Millipore, MAB397), anti-GluA3^{44 (link)}, anti-PSD-95 (Santa Cruz Biotechnology, SC32290), anti-D1R (Frontier Institute, Japan, MSFR101030), and anti-D2R (Frontier Institute, Japan, MSFR101060) were used for western blotting.