Immunoprecipitation of p-CaMKIIα and GluN2B

Immunoprecipitation was used to detect the interaction between p-CaMKIIα and GluN2B. Immunoprecipitation from the extrasynaptic fractions of the hippocampus was performed with rabbit NMDAR2B (2–3 μg) antibody [24 (link)]. Briefly, the extrasynaptic fractions of the hippocampus were obtained as described above. The extracts were precleared by adding nonspecific control immunoglobulin G (1 μg) and 20 μl of Protein G Sepharose (Sigma, USA). The supernatant was collected and incubated with nonspecific IgG (2 μg) or rabbit anti-NMDAR2B (2 μg; Abcam, Cambridge, UK) overnight at 4 °C. The next day, they were mixed with the addition of 40 μl of Protein G Sepharose (Sigma, USA) and then rotated slowly for 4 h at 4 °C. After that, the beads were washed three times in buffer A (in mM): 150 NaCl, 50 Tris-HCl, 0.1% Triton X-100, and 1 EDTA. Then, the beads were washed three times in buffer B (in mM): 300 NaCl, 50 Tris-HCl, 0.1% Triton X-100, and 1 EDTA. Finally, the beads were denatured in SDS buffer and separated by SDS-PAGE.