Glun2a

The methods used to produce the GST fusion proteins in bacteria have been certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
previously reported (Anzai et al., 2002; Anzai et al., 2004) . GST-pull-down assays were carried out using a GST Protein Interaction Pull-Down Kit (Pierce) according to the manufacturer's instructions. Briefly, the bacterial lysate containing the fusion proteins was sonicated and centrifuged, and the supernatant was used for protein binding assays. In vitro translation was facilitated using a plasmid carrying full-length GluN2A, GluN2B or GluN1 (Addgene) that was transfected into HEK293T cells. The translated total proteins obtained from cells and the GST fusion proteins were incubated together with GST glutathione agarose resin. The protein complexes were then eluted by glutathione and analyzed by Western blotting using GST, GFP, GluN2A, GluN2B or GluN1 antibodies.
For co-immunoprecipitation assays, the extracts contained in the crude synaptosomal fraction obtained from adult mouse hippocampal tissues were prepared with anti-GluN2A, anti-GluN2B, anti-GluN1 or anti-FRMPD2 antibodies or IgG (rabbit, Abcam) (Control) at 4°C for 12 h before they were incubated with protein A/G agarose beads (Beyotime). The immunoprecipitated mixture was immunoblotted with GluN2A, GluN2B, GluN1 or FRMPD2 antibodies.