Hnf1α

Cultured cells were collected with cell lysis buffer (Beyotime, Shanghai, China). Protein samples were separated by precast NuPAGE Novex 4–12 % (w/v) Bis–Tris gels (Life technologies, Carlsbad, CA, USA) and then transferred onto nitrocellulose membrane using the iBlotTM dry blotting system as described by the manufacturer (Invitrogen, Carlsbad, CA, USA). Membranes were blocked in TBST buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 0.1 % tween 20) containing 5 % milk for 1 h at room temperature and incubated with primary antibodies specific for LDLR (Biovision, Mountain View, CA), PCSK9 (Cayman Chemicals, MI), HNF1α (Cell Signaling) and GAPDH (Abcam) overnight at 4 °C, and then incubated with a secondary antibody conjugated with horseradish peroxidase (HRP) for 2 h at room temperature. Blots were developed using chemoluminescence (ECL, Thermo Fisher Scientific, Waltham, MA, USA) on FluorChem M image system.

To analyze the protein expression levels of LDLR, PCSK9, SREBP2, and HNF-1α, HepG2 cells were seeded and treated as described above, and the cells were washed with cold DPBS, harvested, and centrifuged (1200 rpm, 3 min). The collected pellet was lysed with the RIPA buffer (Elpis Biotech, Inc.) containing protease and phosphatase inhibitors (Roche, Basel, Switzerland). The lysates containing equal amounts of protein, were then loaded on Bis-Tris gels for electrophoresis (10%), before being transferred to nitrocellulose membranes. The blots were blocked in 5% skim milk solution for 1 h and probed with specific antibodies (PCSK9 (MBL Life Science, Woburn, MA, USA), LDLR (Biovision Inc., Milpitas, CA, USA), HNF-1α (Cell Signaling Technology, Inc., Danvers, MA, USA), SREBP2 (Cayman Chemical, Ann Arbor, MI, USA), and β-actin (Bethyl Laboratories Inc., Montgomery, TX, USA)]. After washing three times with tris-buffered saline (TBS) with Tween 20, the blots were incubated with peroxidase-conjugated purified goat anti-rabbit IgG (Enzo Life Sciences, Inc., Farmingdale, NY, USA) for 1 h. After an additional wash with TBS with Tween 20, the proteins were detected using chemiluminescence reagent (Pierce Biotechnology, Rockford, IL, USA). The chemiluminescent signal was analyzed using the Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA).