Fig. 2.

Functional analysis of the extracellular ABCG2 residues. Residues in the extracellular ABCG2 regions were subjected to mutational analysis, followed by functional expression in HEK293 cells. a Immunodetection of ABCG2 variants after transient transfection into HEK293 cells for 2 days is shown. β-Actin was used as a loading control. b Odyssey-based quantification of immunoblots from several independent experiments (n = 3–14). ABCG2 signals were individually normalized to β-actin and shown as the percentage of the WT control. Cell-free extracts from HEK293 cells were prepared from the same experiment; immunoblots from different gels are separated by white spaces. c Mitoxantrone efflux is shown for HEK293 cells transfected with ABCG2 variants. Mitoxantrone accumulation was quantified in the presence and absence of Ko143 inhibitor after incubation for 20 min at 37 °C. Ko143-sensitive mitoxantrone efflux is given as percentage efflux activity relative to WT control. Data are from several independent experiments (n = 3–9). All results are represented as means ± SEM; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.1. Gray dots represent values from each independent experiment. d Membrane localization of GFP-tagged ABCG2 mutants was detected by fluorescence microscopy in the GFP channel (green) as described in Methods. Nuclei were stained with DAPI (blue). Microscopy data are from representative duplicate experiments. Scale bars in microscopy images correspond to 20 µm.