Fig. 4.

Diagnosis of PFIC subtype using liver specimens and in vitro mutagenesis study in the patients with normal-GGT PFIC.

(a) Expression of ATP8B1 and BSEP in livers of patients with normal-GGT PFIC. Membrane fractions were prepared from liver specimens of the patients, and 5 μg (left panel) and 20 μg (right panel) of the specimens were subjected to immunoblotting. The signal intensity of ATP8B1 and BSEP relative to that of NaK is presented below each panel. Control 1; HBV, Control 2; HCV, Control 3; HCV, Control 4; Alagille syndrome. a.u., arbitrary unit; BQL, below the limit of quantitation; NaK, Na⁺, K⁺-ATPase α1 subunit. (b–d) Characterization of missense mutations of ATP8B1 in patients with PFIC1 and PFIC1-like disease. CHO-K1 cells were transfected with pShuttle-ATP8B1^WT–FLAG, ATP8B1^Triple–FLAG, ATP8B1^C306R–FLAG, or ATP8B1^E981K–FLAG together with pShuttle-HA–CDC50A and subjected to cell surface biotinylation (b), immunocytochemistry (c), and a flippase assay (d, e). CHO-K1 cells transfected with the corresponding empty vector were employed as control cells. A representative result of at least two independent experiments is shown. (b) Protein expression. Upper, the cells were biotinylated, lysed, precipitated with streptavidin, and then analyzed by immunoblotting. Lower, quantification of the amount of the mutated ATP8B1 in the cell surface fraction. The band intensity shown in the upper panel was quantified as described in the Supporting information. Each bar represents the mean ± SEM of triplicate determinations. *, P < 0.05. (c) Cellular localization. The cells were subjected to immunocytochemistry and analyzed by confocal immunofluorescence microscopy. Yellow in the merged images indicates colocalization of FLAG and NaK. Scale bar: 10 μm. (d, e) Determination of flippase activity. The cells were incubated with NBD-PC at 15 °C for the indicated time (d) or 15 min (e) and then with fatty acid-free BSA on ice for 5 min. The residual fluorescence intensity associated with the cells was determined by flow cytometry. Each bar represents the mean ± SEM of triplicate determinations. ***, P < 0.001. BQL, below the limit of quantitation; EV, empty vector; WT, wild type.