Glun2b

Western blotting was performed according to methods established in our laboratory^{46 (link)}. Hippocampus was removed and homogenized on ice with RIPA buffer (Bi Yuntian). The samples were separated by SDS-PAGE, transferred onto nitrocellulose membranes and then covered by 5% non-fat milk. After washings by Tween-TBS, the membranes were detected by primary antibodies overnight, and then by secondary antibodies conjugated to IRDyeTM (800CW) for 1 h. The visual blots were acquired from the Odyssey Infrared Imaging System (Licor biosciences, Lincoln, NE, USA). The full length blots are presented in Supplementary Information. The primary antibodies are displayed as follows: CREB (cell signaling, 1:1000), p-CREB (cell signaling, 1:1000), BDNF (SANTA CRUZ, 1:1000), GluN2A (abcam, 1:1000), GluN2B (abcam, 1:1000), GluR1 (Millipore, 1:500), GluR2 (Millipore, 1:500), PSD95 (cell signaling, 1:1000), anti-Synaptotagmin1 (abcam, 1:2000), anti-Synapsin1 (Millipore, 1:1000), PT231 (SAB, 1:500), PP1 (Millipore, 1:200), HT7 (Thermo, 1:500), β-actin (abcam, 1:1000), acetylated H3 (Millipore, 1:1000), acetylated H3K14 (Millipore, 1:1000), acetylated H3K9 (Millipore, 1:1000), Histone 3 (abcam, 1:1000).

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.

Transfected neurons were treated at DIV10 with rWnt5a for 1 h and then washed thrice with PBS Ca²⁺/Mg²⁺ (100 µM/1 mM), fixed with a freshly prepared solution of 4% PFA-sucrose in PBS for 20 min and permeabilized for 5 min with 0.2% Triton X-100 in PBS. Neurons were incubated with primary antibody (GluN2B Abcam, 1:700) overnight at 4 °C in a wet chamber. Secondary antibody coupled to 555 nm fluorophore was applied at a 1:500 dilution for 30 min at 37 °C.