Hnf1α

The protein level was determined, according to the method of Tan et al. [31 (link)]. The antibodies used in this study included: PPARα (Absin, Shanghai, China), CPT1 (Proteintech, USA), FXR (Bioss, Beijing, China), HNF1α (Abcam, Cambridge, UK), GRP78 (Cell Signaling Technology, CST, Danvers, MA, USA), p-PERK (Bioss, Beijing, China), p-EIF2α (CST, Danvers, MA, USA), JNK (CST, Danvers, MA, USA), p-JNK (CST, Danvers, MA, USA), P38 (CST, Danvers, MA, USA), p-P38 (CST, Danvers, MA, USA), ERK(CST, Danvers, MA, USA), p-ERK(CST, Danvers, MA, USA), c-Jun (CST, Danvers, MA, USA), p-c-Jun (CST, Danvers, MA, USA), GAPDH (ZSGB-Bio, Beijing, China), FLAG (CST, Danvers, MA, USA), HA (CST, Danvers, MA, USA) and GFP (CST, Danvers, MA, USA). An electrochemiluminescence kit (Beyotime, Beijing, China) was used to visualize the immunoreactive protein, which was then scanned with an Epson Perfection V33 scanner.

The proteins were prepared by homogenizing HepG2 cells in RIPA buffer ((1% v/v Triton X-100, 0.1% w/v sodium dodecyl sulfate, 0.5% w/v sodium deoxycholate, 10 mM sodium phosphate, pH 7.5, 5 mM EDTA, 5 mM EGTA, 100 mM sodium chloride, 1 mM PMSF, 20 M leupeptin, 20 mM methionine, and 1 mM cysteine)) containing a protease inhibitor cocktail on ice. The proteins were resolved on a 5% stacking, 8% running gel and transferred onto a nitrocellulose membrane. The membranes were blocked overnight with 5% nonfat dry milk in PBS with 0.1% Tween 20 and probed with primary antibodies overnight at 4 °C, followed by incubation with secondary antibodies for 90 min at room temperature. The membrane-bound antibodies were visualized using horseradish peroxidase-conjugated secondary antibodies (1:12,000) and the Odyssey Infrared Imaging System (Li-Cor Bioscience, Bad Homburg, Germany). Normalization was performed by blotting the same samples with an antibody against GAPDH. The primary antibodies were: PCSK9 (Abcam), LDLR (Abcam) and HNF1α (Abcam), each used at a dilution of 1:1000.