MDCK-ABCG2 cells were permeabilized to allow the titration of intracellular nucleotide concentrations. Dose-response curves of 5D3 binding with increasing concentrations of ATP/Mg2+ (panels (A), and (B)), ATP in the presence of EDTA (panels (C) and (D)), AMP-PNP/Mg2+ (panels (G) and (H)), or ADP/Mg2+ (panels (I) and (J)) were obtained in the presence or absence of Vi (left panels) or BeFx (right panels). Samples were pre-treated with nucleotides at 37°C for 10 min, then further incubated with 5 µg/ml 5D3-A647 at 37°C for 20 min except for (panels (E) and (F)), where all the treatments were carried out on ice. In case of nucleotide trapping, permeabilized cells were co-treated with nucleotides and BeFx or Vi at 37°C for 30 min, then un-trapped nucleotides were washed out and 5D3 labeling was carried out on ice for 45 min. Representative curves are shown from three to five independent experiments. The small inserts in panel (A) depict the IF and OF conformations of ABCG2. IF, inward-facing; OF, outward-facing.