Figure 4. MTS-verapamil labelling of mutant L65C is inhibited by verapamil.
BHK cells stably expressing His-tagged mutant L65C were solubilized with n-dodecyl-β-D-maltoside. Insoluble material was removed by centrifugation. Equivalent amounts of supernatant were incubated for 10 min at 20 °C in the presence of no drug (No Drug), 2 mM verapamil (Ver) or 3 mM Rhodamine B (Rhod). The samples were then incubated with 0.1 mM MTS-verapamil for 10 min at 20 °C. His-tagged P-gps were isolated by nickel-chelate chromatography. Equivalent amounts of the isolated P-gps were mixed with lipid and assayed for ATP activity in the absence of drug substrate. The activities are expressed relative to a sample treated with MTS-verapamil in the absence of drug substrate. Each value is the average of three different experiments.