Characterization of c.386 G > A (p.C129Y) and c.1460 G > A (p.R487H) mutations in ABCB11. HepG2 cells (a,b) and HEK293T cells (c–e) were transfected with pShuttle-HA-BSEPWT, HA-BSEPC129Y, HA-BSEPR487H, or a corresponding EV. (a,b) Expression and cellular localization of HA-BSEPC129Y and HA-BSEPR487H in HepG2 cells. The cells were immunostained and analyzed through confocal immunofluorescence microscopy. The cellular outline and bile canaliculi were visualized using alexa488-phalloidin. A representative image is shown in (a). Yellow in the merged images indicates colocalization. Scale bar: 10 μm. An analysis of the percentage of cells with each form of HA-BSEP at the bile canaliculus is shown in (b). A total of 30–40 cells immunostained using an anti-HA antibody were analyzed in each coverslip. (c–e) Expression and transport function of HA-BSEPC129Y and HA-BSEPR487H in HEK293T cells. Membrane vesicles prepared from the cells were analyzed through a capillary-based immunoassay (c) and subjected to a transport assay with 0.8 μM [3H]-TC. ATP-dependent uptake of [3H]-TC for 2 min was measured using each batch of membrane vesicles (d). Transport activity of [3H]-TC by each form of HA-BSEP (e) was calculated by normalizing the transport values in (d) by the HA-BSEP expression level determined in (c). In (a–e), a representative result of two independent experiments is shown. Bars represent the mean ± SEM of each experiment in triplicate. **P < 0.01; ***P < 0.001; BDL, below detection limits because of low expression levels; EV, empty vector.