We made a fusion gene that expresses the entire 561 amino acids of the N-terminal extracellular domain of human GluN1 (NR1) (UniProtKB - Q05586), including the amino terminal domain (ATD), followed by the Myc tag, a 6XHIS tag, a TEV protease cleavage site, and the transmembrane domain of the human platelet-derived growth factor receptor (PDGFR) (Additional file 1 , Genbank Accession #) [13 (link), 15 (link)]. The gene was synthesized and inserted into the retroviral vector, pBabe puro by Genscript (Piscataway, NJ) [16 (link)]. Amphotropic retroviruses were produced in 293T cells following standard protocols, except that X-tremeGENE 9 DNA Transfection Reagent was used (354,087, Roche, Germany), and the cells were cultured in Advanced DMEM, 1% IFS, penicillin/streptomycin (Invitrogen, Carlsbad, CA) [17 (link)].
The retroviral supernatant was used to transduce 293T cells (2.5 X106 in a 10 cm dish), with 4 μg/ml polybrene (TR1003G, Thermo Fisher Scientific, Waltham, MA), for 6 h. 48 h later, cells were selected with 1 μg/ml puromycin (P9620, Sigma-Aldrich, St. Louis, MO). One week later, expressing cells were isolated by FACS staining with the murine anti-NR1 APC mAb (orb149996, Biorbyt, San Francisco, CA) on the BD FACSCanto II (Becton Dickson, Franklin Lakes, NJ). Four rounds of FACS over 4 weeks were performed to isolate a stable, homogeneous population of cells (293T-ATD).
The retroviral supernatant was used to transduce 293T cells (2.5 X106 in a 10 cm dish), with 4 μg/ml polybrene (TR1003G, Thermo Fisher Scientific, Waltham, MA), for 6 h. 48 h later, cells were selected with 1 μg/ml puromycin (P9620, Sigma-Aldrich, St. Louis, MO). One week later, expressing cells were isolated by FACS staining with the murine anti-NR1 APC mAb (orb149996, Biorbyt, San Francisco, CA) on the BD FACSCanto II (Becton Dickson, Franklin Lakes, NJ). Four rounds of FACS over 4 weeks were performed to isolate a stable, homogeneous population of cells (293T-ATD).