Surface Protein Biotinylation in HEK293 Cells

HEK293 cells were transfected with human cDNA, and surface protein biotinylation was performed as described previously [67 (link)]. Briefly, 24 hours after transfection, cells were incubated with 1 mg/ml Sulfo-NHS-biotin for 20 min on ice, and then the biotin was quenched with 50 mM glycine. After cell lysis, protein concentrations were determined by the Bradford assay, and equal amounts of protein from each sample were added to Neutravidin beads (ThermoFisher) and rotated for 2 hr at 4°C. Total protein and pulled-down surface protein fractions were separated by SDS-PAGE, and immunoblotted using the following antibodies: mouse anti-GluN1 (BD Pharmingen, San Jose, CA, USA), rabbit anti-GluN2A (Millipore, AB1557), mouse anti-GluN2B (Alomone Labs, Jerusalem, Israel), mouse anti-transferrin receptor (Sigma), and mouse anti-tubulin. Transferrin receptor levels were used to assess whether biotin labeling was consistent across conditions. Tubulin levels were used as a loading control for total protein samples and to ensure only surface proteins were in biotinylated fractions. Chemiluminescence signals were detected with film, imaged with a Bio Rad Gel Imager, and quantified using ImageJ.