HEK293T cell culture and transfection

HEK293T cells (Greenberg lab, originally from ATCC) were maintained in DMEM (Invitrogen), 10% fetal bovine serum (Atlanta Biologicals), 1% penicillin–streptomycin (Invitrogen), and 1% glutamine (Invitrogen). Cells were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol or with calcium phosphate⁶⁵. Briefly, the pH of 2X HeBS (274 mM NaCl, 10 mM KCl, 1.4 mM Na₂HPO₄·7H₂O, 15 mM d-glucose, 42 mM HEPES) was adjusted by NaOH to yield pHs ranging from 7.03, 7.05, 7.07, 7.09, 7.11, to 7.14. 2X HeBS was filter-sterilized, aliquoted and stored at 4 °C. Each pH was tested to determine which provided the best transfection efficiency. To prepare transfection mixture, 70 µL of HEPES-buffered dH₂O (2.5 mM HEPES) was added to an Eppendorf tube, 8.65 µL of 2.5 M CaCl₂ was added to the bottom of the HEPES-containing tube, then plasmid DNA was added on top of the CaCl₂–HEPES mixture and mixed by adding 86.5 µL of the most pH effective 2X HeBS. Precipitation was initiated by bubbling the transfection mixture with a pipette 8–10 times. The mixture was immediately added to HEK cells (one transfection mixture per one well of a six-well plate) dropwise. Transfected cells were immunostained, imaged or lysed 16–24 h after transfection. Myc-GluN1, GluN2B, FLAG-EphB2, and EGFP constructs were co-transfected for HEK293T PLA experiments. 50 µM APV (Tocris Bioscience) and 10 µM MK801 (Tocris Bioscience) were added after transfection to prevent excitotoxicity.