Blue native polyacrylamide gel electrophoresis (BN-PAGE)

BN-PAGE was performed using the NativePAGE Bis-Tris Gel System (Thermo Fisher Scientific, Waltham, MA). Briefly, cells were harvested, washed with HBS and solubilized in NativePAGE Sample Buffer supplemented with 10% glycerol, 1% DDM (n-dodecyl-β-D-maltoside), 1X protease inhibitor cocktail and 100 μg/ml DNaseI. The samples were incubated in the solubilization buffer on ice for 30 min, centrifuged (13000g at 4°C, 1 hr), and the resulting supernatants were either untreated or treated with 4M urea to denature protein complexes. Samples were then fractionated on precast 4–16% BN gradient gels (Thermo Fischer Scientific). After electrophoresis, proteins were transferred to PVDF membranes (Millipore Corp., Bedford, MA) and detected by our standard immunoblotting protocol with anti-TfR or anti-VSG221.