Fig. 7. Ligand-induced internalization and recycling of ALCAM/CD166 molecules. (A) A2774 cells were surface-labeled with NHS-SS-biotin, incubated for 30 minutes at 37°C with or without the anti-ALCAM/CD166 mAb 3A6, treated with the reducing agent GSH, lysed and immunoprecipitated with I/F8 scFv coupled to Sepharose beads. Residual biotin-labeled molecules identify internalized proteins. (B) The kinetics of 3A6 mAb-triggered ALCAM/CD166 internalization was evaluated at different time points prior to GSH treatment and lysis. Optimal receptor internalization is at 40 minutes. The efficiency of surface NHS-SS-biotin stripping by GSH was evaluated at different time points on 3A6-treated cells maintained at 4°C. (C) To analyze receptor recycling, biotin-labeled and 3A6-treated A2774 cells were allowed to internalize ALCAM/CD166 for 40 minutes and were treated with GSH. Cells were then re-incubated at 37°C for 20 minutes to chase internalized receptors, and treated with (+) or without (–) the reducing agent to remove the label from molecules re-expressed to the cell surface. Cell lysis and I/F8-sepharose bead immunoprecipitation followed. Internalized ALCAM/CD166 recycles to the cell surface. (D) Kinetics of recycling. NHS-SS-biotin-labeled internalized receptors were chased by re-incubation at 37°C for different time points (t) before the second GSH treatment and immunoprecipitation. Immunoprecipitates were resolved by SDS-PAGE and revealed by western blot analysis with HRP-streptavidin and chemiluminescence.