Cyp7a1

Formaldehyde-fixed (10%) and paraffin-embedded liver tissue sections were stained with hematoxylin and eosin. Four-micrometer-thick sections were cut from paraffin block and coated on a glass slide. Immunohistochemistry for HNF4α, NTCP, and Cyp7a1 (Abcam, Cambridge, UK) expressions (Abcam, Cambridge, UK) was performed. A single-blinded pathologist evaluated histological characteristics, intensity, extent, and immune-reactive scores of immune-stained sections.

Total proteins from liver and ileum tissues were extracted as previously described (62 (link)). Total proteins (40 μg per lane) were separated by 10% SDS-PAGE and then electrotransferred to nitrocellulose membranes. The membranes were incubated with primary antibodies against FXR (NR1H4, 1:1,000; Abcam, USA), SREBP2 (1:1,000; Abcam, USA), CYP7A1 (1:1,000; Abcam, USA), CYP8B1 (1:1,000; Abcam, USA), CYP7B1 (1:1,000; Proteintech Group, USA), CYP27A1 (1:1,000; Proteintech Group, USA), BSEP (1:200; Santa Cruz Biotechnology, USA), and β-actin (1:1,000; Cell Signaling Technology, USA) overnight at 4°C. After washing with 0.1% Tween 20–phosphate buffer solution (PBST) three times, the membranes were incubated with goat anti-mouse or goat anti-rabbit IRDye 700 or 800 calcofluor white (CW)-labeled secondary antibodies for 1 h at 37°C and imaged with an Odyssey infrared scanner (LI-COR, Lincoln, NE, USA). Quantification was performed using the LI-COR software Image Studio.