Anti glun1

Protein extracts were separated by 4–20% Mini-PROTEAN TGX Gels (Bio-Rad Laboratories, Temse, Belgium, cat#561091) and transferred to nitrocellulose membranes (GE Healthcare, Milan, Italy). Blots were immunostained overnight at 4 °C with the following primary antibodies: Purified anti-β-Amyloid (Biolegend, San Diego, CA, USA, cat#SIG-3920), anti-GluN2A (Merck Millipore, Hoeilaart, Belgium, cat#07-632), anti-GluN1 (Merck Millipore, Hoeilaart, Belgium, cat#05-432) and anti-PSD95 (Merck Millipore, Hoeilaart, Belgium, cat#MAB1598). After washing, the membranes were incubated for 2 h at room temperature with the appropriate secondary antibody (anti-mouse, GE Healthcare, Milan, Italy, cat#NXA931; anti-rabbit, cat#NA934V). Following secondary antibody incubations, membranes were washed and finally incubated with ECL detection reagent (Amersham, UK, cat#RPN2232) for 5 min.
Immunocomplexes were visualised by chemiluminescence using the LI-COR Biosciences Imaging System (LI-COR Biosciences, Bad Homburg vor der Höhe, Germany) and analysed using the Image Studio Lite software 5.2 (LI-COR Biosciences, Bad Homburg vor der Höhe, Germany).

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.

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.