123 retention. The relative value of the Rho 123 retention was calculated by dividing the Rho 123 for each measure Ma Because identifies obtained in the absence of FG020326 in ABCB1 KBv200 expressing cells. 2.11. Photoaffinit Tsmarkierung the ABCB1 azidopine PCI-24781 KBv200 cell membranes were prepared, and the experiment was performed as described. The membranes were blocked for 25 min with azidopine FG020326 in the dark, by incubation with 0.6 M Followed similar incubated. After UV irradiation for 2 minutes, were photolabeled membranes by SDS-PAGE on a gel 8, followed by subjected to fluorography. The presence of ABCB1 was best by Western blot using the monoclonal Rpers C219 and ECL detection apparatus CONFIRMS. 2.12.
ATPase assay ABCB1 ABCB1 efflux function, the hydrolysis of ATP, stimulated in the presence of substrates ABCB1 GSK256066 is linked. ATPase activity t ABCB1 of Vi in membrane vesicles of High Five insect cells was measured as described above. Membrane vesicles were ATPase assay buffer was incubated with or without 0.3 mM vanadate for 5 to 37 min, then with various concentrations of up to 37 for 3 FG020326 incubated. The ATPase reaction was induced by the addition of 5 mM ATP, Mg, and the total volume was 0.1 ml After incubation at 37 for 20 minutes, the reactions by charging 0.1 ml 5 SDS stopped. The Pi ver Ffentlicht was measured as described. 2.11. Expression of ABCB1 After treatment for 48 h were harvested FG020326 KBv200 cells rinsed twice with PBS.
Cell extracts were prepared with buffer for 30 min with gentle rocking and clarified Rt by centrifugation at 12,000 g for 15 min at 4 Identical amounts Cell lysates were boiled for 10 min and gel St by sodium dodecyl sulfate gel electrophoresis and electrophoretically transferred to PVDF membranes polyacrylamide. After in Blockierungsl Solution containing 5 skim milk in TBST buffer, 150 mM NaCl, Tween 20, and 0.1 were incubated for 2 h at 4, the membranes with primary Rem Antique Body incubated diluted fa be appropriate. The expression of actin was used as the load embroidered. The membranes were then incubated for one with HRP-conjugated secondary Rem Antique Body incubated 1:1000 dilution. The proteins Were verst by the detection system Proven markets chemiluminescence. Protein expression was quantified by Scion Image software. 2.12.
Location FG020326 in cells by confocal fluorescence FG020326 an original composition. KBv200 cells were incubated at 37 for 5 h FG020326 5,000,000. The cells were collected after treatment with trypsin and washed twice with PBS. Subsequently End, the cells were a monoclonal Body, the conjugate for ABCB1 directly to PE 0.5 h at room temperature. After three washes with PBS, the cells were at Objekttr Willingly mounted and observed under a confocal microscope, and digital images were recorded. 2.13. Statistical analysis All experiments were repeated at least 3 times, and the differences were det