Labeling, Purification and SDS-PAGE of NMDA Receptor Complexes

For expression analysis, surface receptors were labeled with Pierce™ Premium Grade Sulfo NHS-SS-Biotin (Thermofisher, Waltham, United States) and purified using Streptavidin High Performance Spintrap™ (Sigma-Aldrich, St. Louis, United States). If samples were also treated with DTT, they were incubated with 100 mM DTT (Stocksolution 2 mM DTT in ddH₂O) for 20 min at 56°C. Isolated surface proteins were separated on linear 10% SDS-PAGE gels. PVDF membranes and the Trans-Blot®Turbo™Blotting System (Biorad, Hercules, United States) were used for Western blot analysis. Two different antibodies were used as primary antibodies, firstly the GluN1-CTD was addressed with an antibody from Merck Millipore (Darmstadt, Germany) diluted 1:1,000 in TBS, and secondly the GluN1-NTD epitope was addressed with an antibody from Alomone Labs (Jerusalem, Israel) 1:500 in TBS. A horseradish peroxidase-labeled secondary antibody (1:20,000 in TBS) detecting mouse or rabbit IgG was used. Immunoreactive bands were visualized with the Pierce™ ECL Western Blotting Substrate (Thermofisher, Waltham, United States) using the ChemiDoc MP Imaging System (Biorad, Hercules, United States). Metabolic labeling with (35S) methionine (0.2 Mbq per oocyte; >40 TBq/mmol, Amersham Biosciences) was performed as previously described (Schüler et al., 2008). Purification of C-terminal His6-tag-labeled GluN1 and GluN1^ΔM4 by Ni²⁺-NTA agarose (Qiagen) chromatography was performed as in (Madry et al., 2007a). (35S)-Methionine-labeled protein samples were solubilized in SDS sample buffer containing 20 mM dithiothreitol and electrophoresed in parallel with molecular mass markers (SeeBlue^® Plus2 Pre-Stained Standard, Invitrogen) on 8% Tricine-SDS polyacrylamide gels. Gels were blotted, fixed, dried, and exposed to BioMax MR films (Kodak, Stuttgart, Germany) at −80°C. The radioactivity of each protein band was quantified using a PhosphorImager (Molecular Dynamics) and analyzed using the ImageQuant software package. Cy5-NHS labeling (Amersham Biosciences) and subsequent SDS-PAGE were performed as described in (Mesic et al., 2016) and scanned with a gel imager (Typhoon 9,400, Amersham Biosciences) as described (Madry et al., 2007a). To distinguish between mature and immature receptor complexes, 10 µl of affinity-purified receptor was incubated in reducing sample buffer (20 mM DTT, 1% (w/v) SDS) with 1% (w/v) octylglucoside containing 5 U endoglycosidase H (Endo H) or peptide: N-glycosidase F (PNGase F; both NEB, Frankfurt, Germany) at 37°C for 1 h, and protein samples were analyzed by SDS-PAGE as described above.