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Fig. 2 | Journal of Biomedical Science

Fig. 2

From: The ESCRT-III molecules regulate the apical targeting of bile salt export pump

Fig. 2

The ESCRT-III subunit CHMP5 co-localizes with BSEP in the subapical compartments of hepatocytes. a The flow chart illustrates the identification of CHMP5 as a BSEP-interacting protein via yeast two-hybrid screening. A positive β-galactosidase assay indicates a polypeptide (CHMP5) interacting with the BSEP bait. Lamin was used as a negative control. b CHMP5 interacts with the polypeptide spanning amino acid 484–558 of human BSEP. The CHMP5-interacting domain of BSEP was mapped via yeast two-hybrid screening. Various BSEP fragments were co-expressed with CHMP5 in competent yeast cells and screened by nutritional depletion and β-galactosidase filter assays. c BSEP was co-immunoprecipitated with CHMP5 from three human hepatoma cell lines, Hep G2, Huh-7, and Mahlavu. Total cell lysate (25 μg) was loaded as an input control; normal mouse immunoglobulins (IgG) were used as a negative control of immunoprecipitation. d Endogenous and mCherry-tagged CHMP5 were co-immunoprecipitated with EGFP-BSEP in the total membrane-protein fraction. Hep G2 cells co-expressing EGFP-BSEP and mCherry-CHMP5 or mCherry were fractionated into cytosolic protein (Cyt) and total membrane-protein (tM) fractions. The tM fraction contains the transmembrane and membrane-associated proteins (referred to Additional file 1: Figure S2). The two fractions were subjected to immunoprecipitation and immunoblotting via the antibodies indicated. e Immunofluorescence staining demonstrates that CHMP5 co-localizes with subapical BSEP in the adult human livers. Cryosection of adult human liver samples was stained for BSEP (green) and CHMP5 (red). The liver cell nuclei were stained with DAPI. f Immunofluorescence assays reveal the co-localization of CHMP5-3HA and FLAG-BSEP in the cytoplasmic vesicles in Bsep knockout mouse livers. CHMP5-3HA and FLAG-BSEP were co-expressed in Bsep knockout mice via hydrodynamic injection. Cryosections of the mouse liver samples were stained by anti-FLAG and anti-HA antibodies for FLAG-BSEP (green) and CHMP5-3HA (red), respectively. The liver cell nuclei were stained with DAPI. Arrows indicate the subapical vesicles with both FLAG-BSEP and CHMP5-3HA signals

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