Biotin-based assays were performed as described previously10 (link),13 (link). MDA-MB-231 or PC-3 cells were grown in 10% FBS-containing medium on 6 cm dishes to 80% confluence. The cells were placed on ice and washed once with cold PBS. Cell surface proteins were labelled with 0.5 mg/mL of EZ-link cleavable sulfo-NHS-SS-biotin (#21331; Thermo Scientific) in Hanks' balanced salt solution (H9269; Sigma) for 30 min at 4°C. Unbound biotin was washed away with cold medium, and prewarmed 10% serum-containing medium was added to cells. The biotin-labelled surface proteins were allowed to internalize at 37°C for the indicated time-points, after which the cells were placed quickly back on ice with cold medium. In the case of recycling, the cells were incubated at 37°C for 30 min to allow the internalization of all biotin-labelled integrins. The remaining biotin at the cell surface after internalization was removed with 60 mm MesNa (63705; sodium 2-mercaptoethanesulfonate: Fluka) in MesNa buffer (50 mm Tris–HCl, pH 8.6, 100 mm NaCl) for 30 min at 4°C, followed by quenching with 100 mm iodoacetamide (Sigma) for 15 min on ice. To detect the total amount of surface biotinylation, one of the cell dishes was left on ice after biotin labelling followed by treatment without reducing MesNa. Cells were washed with PBS and in the case of recycling, the cells were incubated again at 37°C for the indicated time-points followed by reduction and quenching of the recycled biotin-labelled surface molecules as above. The cells were lysed by scraping in lysis buffer [1.5% octylglucoside, 1% NP-40, 0.5% BSA, 1 mm EDTA with phosphatase and protease inhibitor coctails (Roche)] and incubation at 4°C for 20 min. Cell extracts were cleared by centrifugation (16 000 × g, 10 min, 4°C), and the biotinylated integrins were immunoprecipitated from the supernatant with 2 ng/μL of anti-β1 integrin antibody (CD29 K20; Custom Design Service, Beckman Coulter) and protein G sepharose beads (17-0618-01; GE Healthcare). Internalized integrins were detected from immunoblots with horseradish peroxidase (HRP)-conjugated anti-biotin antibody (#7075; Cell Signaling Technology), and after stripping the immunoblot, the total amount of immunoprecipitated integrins was detected with anti-β1 integrin antibody (MAB2252, Chemicon, or 610468, BD Transduction Laboratories). Enhanced chemiluminescence-detected biotin and β1 integrin signals were quantified as integrated densities of the protein bands with ImageJ (version 1.43u), and each biotin signal was normalized to the corresponding integrin signal. In the case of internalization, the relative amounts of biotin-labelled integrins inside the cell were normalized to the biotin signal of all surface labelled integrins, while in the case of recycling, the relative biotin signals were normalized to the corresponding biotin signal of all internalized integrins. The decreasing amount of biotin-labelled integrins inside the cell was inverted to represent the increase of β1 integrin recycling. The ELISA-based endocytosis assays were performed as described above. To determine the amount of internalized, biotinylated integrins, the cell lysate was incubated in a 96-well plate using the well-coating anti-β1 integrin antibody K20 and an HRP-coupled anti-biotin antibody for ELISA detection.
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