ATPase Assays.

The assay was conducted to measure the amount of inorganic phosphate released from the ABCB4-mediated ATP hydrolysis reaction at various conditions (35). Nanodisc-reconstituted wild-type ABCB4 in HBS was used in the concentration range of 0.01 to 0.03 mg⋅mL⁻¹. Fab-fragments were used in at least threefold excess to ensure saturation. Posaconazole (Sigma) was dissolved in dimethyl sulfoxide (DMSO) and sequentially diluted to 100× working stock solutions. Upon addition of posaconazole, each reaction sample contained 1% DMSO. All ATPase reactions were started by addition of 4 mM ATP and 10 mM MgCl₂, incubated for 0 to 60 min at 37 °C and stopped by adding 6% SDS. Using GraphPad Prism 8, ATPase rates were calculated from linear regressions, and the IC₅₀ value was determined from the sigmoidal interpolation (four parameter logistic equation) of the data points.