Fig. 4. Protein trafficking of OATP1A2 in the overexpressing HEK-293T cells with or without compound C treatment. (A) OATP1A2 internalization process. (Left) OATP1A2 internalization assay was conducted as described in the Materials and Methods. Cells were pretreated with compound C or dimethylsulfoxide (DMSO; 10 μM, 60 minutes, 37°C). Cells were allowed to internalize for 0.5, 1.5, and 3 minutes at 37°C. (Right) Densitometric analysis of the internalized transporter protein as a percentage of the total initially biotinylated OATP1A2 at the cell surface. (B) OATP1A2 membrane-targeting process. (Left) OATP1A2 targeting was evaluated as described in the Materials and Methods. Cells were pretreated with compound C or DMSO (10 μM, 60 minutes, 37°C). Additional biotin labeling was allowed for 5, 10, and 15 minutes at 37°C. (Right) Densitometric analysis of transporter protein that was newly targeted to the cell surface (newly synthesized and previously internalized protein combined) as a percentage of the total initially biotinylated OATP1A2 at the cell surface. (C) OATP1A2 recycling process. (Left) OATP1A2 recycling was assessed as described in the Materials and Methods. Cells were pretreated with compound C or DMSO (10 μM, 60 minutes, 37°C). Cells were allowed to internalize for 1 minute at 37°C, and after the first stripping step, cells were allowed to internalize for 1, 2, or 3 minutes at 37°C. (Right) Densitometric analysis of recycled transporter protein as a percentage of the total initially biotinylated OATP1A2 at the cell surface before initiation of recycling (the protein signal at t = 0 minutes was subtracted from each time point). A representative blot is presented; each experiment was repeated on three occasions. Values are the mean ± S.D. (n = 3). **P < 0.01, different from DMSO-control. NC, vector-transfected HEK-293T cells; SC, stripping control.