Fig. 5

Subcellular localization of wt and mutants ABCB4 in HepG2 and HEK293 cells by immunofluorescence and confocal microscopy. (A) HepG2 cells were transiently transfected with ABCB4-wt or mutants. Forty-eight hours later, cells were fixed and permeabilized, and processed for immunofluorescence using the anti-ABCB4 (P3-II-26) and anti-MRP2 (M2-I-4) monoclonal antibodies, followed by goat anti-IgG2b Alexa Fluor 488- and goat anti-IgG1 594-conjugated secondary antibodies, and visualized by confocal microscopy. yellow denotes colocalization of ABCB4-wt and all mutants with endogenously expressed MRP2 used as a canalicular marker in merged images. Nuclei are stained in blue with Draq5. Transfected cells are indicated by dashed lines. Bile canaliculi are indicated by asterisks. Bars: 10 μm. (B) Localization of ABCB4-wt or the mutants in stably transfected HEK293 cells was assessed by indirect immunofluorescence using anti-ABCB4 antibodies as in (A). Bars: 10 μm.