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. 2017 Oct 23;7:13767. doi: 10.1038/s41598-017-11794-w

Figure 2.

Figure 2

Mutational analysis of the transmission interface. All charged resideus in the predicted TMH2, ICL1 and TMH3 were subjected to mutational changes as indicated. (A) Configuration at the transmission interface of a human ABCG2 monomeric half transporter in two views rotated by 90°, emphasizing the contacts of the transmembrane domain (TMD) with the NBD surface (green); ICL1 (orange); TMHs (transparent white); distal helical parts of TMH2 and TMH3 (yellow); elbow helix (pink); C-terminus (black). Charged residues are indicated as colored balls-and-sticks, positive charges (blue), negative charges (red); not drawn to atomic scale. (B) Immunodetection of ABCG2 variants using the monoclonal mouse anti-ABCG2 (BXP-21) antibody. Mature wild type (WT) ABCG2 migrates to approximately 72 kD, the bands below are immature unglycosylated monomers. β-actin was used as a loading control. (C) Quantified immunoblot using the Odyssey system from several independent experiments (n = 2–12). Mature ABCG2 and β-actin were gated, and ABCG2 signals were individually normalized to β-actin and shown as a percentage of WT. The blots from different gels were divided by the white spaces. (D) Mitoxantrone efflux in transfected HEK293 cells after incubation at 37 °C for 20 min in the presence and absence of Ko143. Ko143–sensitive mitoxantrone efflux is given as percentage relative to WT. Data are from several independent experiments (n = 2–7). (E) Vanadate-sensitive ATPase activity of ABCG2 variants in transfected HEK293 cells. Vanadate-sensitive ATPase activity is represented as a fold change relative to WT. Data are from several independent experiments (n = 8) using three batches of membrane preparations. The ATPase-dead mutants, K86A and K86M served as additional controls. All data are shown as mean and SEM. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.1 vs. empty plasmid transfected HEK293 (mock). (F) Membrane localizations of GFP-tagged ABCG2 variants were visualized by confocal microscopy to detect GFP-tagged ABCG2 variants (green). Nuclear DNA was stained with DAPI (blue). Microscopy data are from duplicate experiments.