Surface proteins labeled with biotin10. Briefly, cells were washed three times with ice-cold PBS and incubated with 1mg/ml Sulfo-NHS-LC-biotin in PBS at 4°C for 20 min. Excess NHS groups were quenched using 100 mM glycine followed by 3 washes with PBS. 1% Nonidet P-40, with protease inhibitor cocktail (Roche) was used to lyse cells and then centrifuged for 10 min at 14,000 rpm at 4 °C. Protein supernatants were mixed with 120 µl of immobilized Neutravidin beads and incubated at 4 °C overnight with gentle rotation. Beads collected by centrifugation were washed three times with lysis buffer and surface proteins labeled with biotin were separated by SDS-PAGE and analyzed by immunoblotting. Blots were cropped to show relevant bands; all full-sized blots are shown in Supplementary Figure S1.
