Ez link cleavable sulfo nhs ss biotin

Biotin-based endocytosis assays were adapted from a validated biochemical method [37 (link)] and carried out as previously described [28 (link)]. FTC-133 cells were allowed to grow in 10% FBS-containing medium in 10 cm dishes until they reached 80% confluency. Cells were then transferred on ice and then washed once with ice-cold PBS. Cell surface proteins were labeled using PBS containing 0.5 mg.mL^-1 EZ-link cleavable sulfo-NHS-SS-biotin (Thermofisher) for 30 min at 4°C. Unbound biotin was removed with cold PBS before addition of pre-warmed 10% FBS-containing medium. Biotin-labeled surface proteins were allowed to internalize for 10 min at 37°C, and then cells were quickly placed back on ice with cold PBS. After internalization, remaining biotin at the cell surface was removed by washing twice with 50 mM glutathione (Sigma-Aldrich) in an appropriate buffer (10 mM EDTA, 75 mM NaCl, 75 mM NaOH, pH 8.0) for 15 min at 4°C. To determine the total amount of surface biotinylation, one dish was kept on ice after biotin labeling and preserved from glutathione treatment. Cells were washed with ice-cold PBS and then lysed by scraping as described above. 350 μg of biotinylated proteins were immunoprecipitated from the supernatant by adding 40 μL of protein G-sepharose beads (GE Healthcare). Internalized integrins were detected by immunoblotting using anti-β1-integrin antibody (M-106).