2.4. Co-immunoprecipitation (Co-IP) and Western blotting (WB)

Mouse liver tissue was washed with cold PBS and then lysed by homogenizing in cell lysis buffer (Cell Signaling Technology). After centrifugation at 14,000 rpm for 15 min at 4 °C, the supernatants were collected, and the protein concentrations were measured using BCA protein assay reagents (Thermo Fisher). Subsequently, Co-IP was performed according to the immunoprecipitation protocol (Thermo Fisher) (thermo_sher.com/immunoprecipitation). Briefly, antibodies were added to the protein G Dynabeads, incubated with rotation for 10 min at room temperature for binding. The beads–antibody complex was then crosslinked using the crosslinking reagent BS3 at room temperature for 30 min. After washing to remove non crosslinked antibodies, the antibody-crosslinked beads were incubated with equal amounts of protein lysates overnight in a cool room. The beads were washed 5 times with 500 μL of IP cell lysis buffer and then eluted using 50 mM glycine. The eluted proteins were separated in Bolt 4–12% Bis-Tris gradient gel (Invitrogen) and transferred onto PVDF membranes (Azure). After blocking with 5% milk, the membranes were probed at 4 °C overnight with various primary antibodies: anti-Foxa2 (Seven Hills, WRAB-1200), FXR (Cell Signaling, mAb #72105), LXR-α (Abcam, ab41902), and histone H3 (Millipore, 04–928) washed with TBST (20 mM Tris, 150 mM NaCl, 0.1% Tween 20; pH 7.6), and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Promega) at room temperature for 1 h. Finally, after washing with TBST, the antibody-bound membranes were treated with enhanced chemiluminescent Western blot detection.