Npc1l1

Cells were plated in a 6-well dish and grown to ∼70% confluence. They were then lysed in RIPA buffer (Sigma) containing a protease inhibitor cocktail. After measuring protein concentration using the Pierce BCA protein assay (Pierce, Rockford, IL), equal amounts (50 µg) of WT- and C282Y-HFE cell lysate protein were loaded on a 10% pre-cast polyacrylamide gel (BioRad). The proteins were separated by electrophoresis, transferred onto a PVDF membrane and probed with the following primary antibodies: CXCL16 and APOE (Abcam), NPC1L1 (Santa Cruz), HMGCoAR (Biovision), and β-Actin (Sigma) which was used as a loading control. Anti-mouse or anti-rabbit secondary antibodies were purchased from GE healthcare. Bands were visualized with SuperSignal West Pico or West Femto reagent (Pierce) using the Fuji image analyzer LAS-3000 (Fuji film). Protein expression was normalized to actin and presented as a percentage of WT control.

Pioglitazone (PIO) was purchased from MSD Pharma (Singapore) Pte. Ltd. Ezetimibe (EZE) was purchased from DEYUAN PHARM Co. Ltd. (Shanghai, China). The antibodies of SREBP-2(sc-13,552), CYP7A1 (sc-293,193), FXR (sc-25,309), LXR (sc-271,064), BSEP (sc-74,500), CCK-AR(sc-514,303) and NPC1L1(sc-166,802) were purchased from Santa Cruz Biotechnology Co. Ltd. (Santa Cruz, CA). The antibodies of HMGCR (ab174830), ABCG5 (ab87116) and ABCG8 (ab126493) were from Abcam Technology Co. Ltd. (Cambridge, UK). The antibodies of ACAT2 (13294S) and PPARγ (2443S) were from Cell Signaling Technology Inc. (Boston, USA). Histone H3, β-actin and the secondary antibodies were from Bioworld Technology Co. Ltd. (Minneapolis, MN, USA). BCA assay kit was from Beyotime (Nanjing China). Amplex Red cholesterol (CH) assay kit was from the Invitrogen (USA). All other chemical or Biological reagents were commercially available.