Figure 3

Effect of telaprevir on photoaffinity labeling of P-gp and BCRP with [¹²⁵I]-IAAP. K562/MDR- or K562/BCRP-vesicles were mixed with the indicated concentrations of telaprevir for 5 min. Vesicles were pre-incubated with 5 nmol/L [¹²⁵I]-IAAP (2200 Ci/mmol) for 10 min, and then illuminated with a UV lamp (365 nm) for 30 min. IAAP-labeled protein was solubilized by Laemmli SDS buffer directly (for the P-gp assay) or after immunoprecipitation (for the BCRP assay), and subjected to SDS-PAGE. Signals were visualized and analyzed with a FLA7000 system. The black arrowheads are P-gp or BCRP. Verapamil and FTC (typical inhibitors of P-gp or BCRP, respectively) were used as controls. The binding of [¹²⁵I]-IAAP with P-gp and BCRP was calculated from standard curve analysis using a FLA7000 Bioimage Analyzer (Fuji Film, Tokyo, Japan) and Multi-Gauge software (Fuji Film).