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FITC-agonist was added for 30 min at 37 C to induce internalization, then removed and washed with cold HBSS to arrest further endocytosis, and cells treated with 500 mM ice-cold sodium 2-sulfanylethanesulfonate (MesNa), a membrane-impermeable reducing agent, in alkaline TNE buffer (100 mM NaCl, 50 mM Tris-HCl, pH 8.6) to cleave BG-SS-biotin specifically from cell surface receptors