Figure 4.

Effect of FG020326 on [³H]-azidopine photoaffinity labeling of ABCB1 (A), the photoaffinity labeling of ABCB1 with [³H]-azidopine was performed with different concentrations of FG020326. The radioactivity incorporated into ABCB1 was determined by exposing the gel to an X-ray film at −70°C; ATPase activity of ABCB1 (B), the Vi-sensitive ATPase activity of ABCB1 in membrane vesicles was determined with different concentrations of FG020326, as described in “Materials and Methods”. The basal ATPase activity (in the absence of FG020326) was subtracted from the activities obtained at the indicated FG020326 concentrations and the graph was fitted by non-linear least-squares regression analysis using GraphPad Prism version 2.0; The expression of ABCB1 (C), equal amounts of total cell lysates were used for each sample and immunoblots were detected with western blot described as “Materials and Methods”. Experiments were repeated at least three independent times, and a representative experiment was shown; FG020326 co-localized with ABCB1 to plasma membrane (D). KBv200 cells were visualized under a confocal microscope and digital images were captured.