Cells were incubated for 40 min at 4°C with EZ-Link Sulfo-NHS-SS-Biotin solution (0.5 mg/ml in PBS; Thermo Fisher Scientific). Surface biotinylation was then quenched with PBS containing 50 mM glycine. To initiate EGFR internalization, cells were incubated in Opti-MEM containing EGF for the specified time periods at 37°C and then cell surface biotin was stripped using MesNa buffer [50 mM 2-mercaptoethanesulfonic acid sodium salt (Sigma-Aldrich)] for 1 hour at 4°C. One sample of cells was left unstripped as a control for surface labeling efficiency. Cells were then lysed with radioimmunoprecipitation assay buffer containing protease inhibitor cocktail and phosphatase inhibitors (100 μM vanandate and 1 μM calyculin A). Biotinylated proteins were then isolated by pull-down using NeutrAvidin Agarose resin (Thermo Fisher Scientific) overnight at 4°C. Samples were then boiled with equal volumes of SDS-PAGE sample buffer and analyzed by immunoblotting. The level of internalized EGFR at different time points was determined as the percentage of difference between non-MesNa stripped cells and stripped cells and corrected for any variations of the total cell lysate protein of each sample.

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