Cell Surface Biotinylation Internalization Assay

Cell surface biotinylation of MEFs and internalization was performed as described (Knorr et al., 2009). Briefly, cells were detached with 7.5 mM EDTA in PBS for 10–15 min at 37°C, washed and resuspended in Hanks’ balanced salt solution (HBSS). 0.5 mg/ml biotinylation reagent Sulfo-NHS-SS-Biotin (Pierce) was added for 30 min on ice before stopping the reaction with cold culture medium (DMEM) supplemented with 50 mM glycine for 60–75 min. After washing with cold HBSS, endocytosis was started by incubating the labeled cells with 37°C warm endocytosis medium (HBSS supplemented with 1% BSA and 1% FCS (EM)) for 2–16 min at 37°C. Endocytosis was stopped by adding ice-cold EM followed by glutathione-stripping of remaining surface biotin with GSH-stripping buffer (50 mM GSH, 100 mM NaCl, 10% FCS at pH 8,5). Cells were lysed in lysis buffer (50 mM TRIS pH 7.5, 100 mM NaCl, 1% TX-100 supplemented with leupeptin, PMSF, E-64, pepstatin, and iodoacetamide) over night at 4°C. After ultracentrifugation for 45 min at 25,000g, internalized biotinylated proteins were isolated from the supernatant with neutravidin-agarose (ThermoFisher) and subjected to immunoblotting.