To assess internalization of integrin, serum-starved WT and CD13KO MEFs or WT KS and CD13 CRISPR KS cells were surface-labeled with NHS-S-Biotin (0.2 mg/ml; Pierce) at 4°C for 30 min. Labeled cells were washed in cold PBS, and internalization was allowed to proceed at 37°C for the times indicated in the figures, at which point the remaining surface biotin was removed by treatment with 20 mM MesNa in 50 mM tris-HCl (pH 8.6) and 100 mM NaCl for 15 min at 4°C. MesNa was quenched by iodoacetamide (20 mM) treatment. Cells were lysed in radioimmunoprecipitation assay lysis buffer containing a protease inhibitor cocktail, and the amount of internalized β1 integrin was quantified. Cell lysate was normalized to total protein concentration, and amount of biotinylated integrin was determined by capture ELISA (20). Briefly, 96-well microtiter plates were coated with 9EG7 (5 μg/ml; MEFs) or 12G10 (KS1767) in 0.05 M sodium carbonate (pH 9.6) at 4°C. Plates were blocked in PBS containing 5% BSA at room temperature for 1 hour. Captured integrin was measured by overnight incubation of cell lysate (100 μg) at 4°C. Plates were washed in PBS/Tween-20 and incubated with streptavidin-HRP for 1 hour at 4°C, and biotinylated integrin was measured by addition of a chromogenic agent ortho-phenylenediamine. Internalized integrin was represented as a percentage of the initial surface integrin pool measured in cells without MesNa treatment at time 0 min.

To determine integrin recycling, cells were surface biotin–labeled, and internalization was allowed to proceed during a period of unperturbed incubation for 30 min. Following removal with MesNa, cells were chased in complete medium at 37°C for the times indicated in the figures. At each harvest point, biotin was eliminated from the surface by a second round of MesNa treatment to exclude integrins that had recycled back to the surface, and the level of biotinylated integrin remaining in the cells (internalized) was measured in cell lysates by capture ELISA. The amount of recycled integrin is based on the assumption that (% internal + % recycled = 100%) and calculated as the difference between time 0 min (maximal internalization) and the relative amount of integrin remaining in the cell at each subsequent chase point or 100% – % internalized at chase (internal at time x/max internal time 0 × 100) = % recycled.

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