Polyvinylidene difluoride membranes

The GCs were lysed in RIPA buffer (Beyotime, Shanghai, China) containing 1% (v/v) PMSF (Boster, Wuhan, China) for 30 min on ice. The lysates were centrifuged at 13,000 rpm for 10 min at 4 °C. Protein concentrations were quantified using a BCA protein assay kit (Boster, Wuhan, China). The proteins (10 µg) were separated using 8% or 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Boster, Wuhan, China). The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline (TBS) for 1 h at room temperature. Then, the membranes were incubated with rabbit polyclonal primary antibodies against NICD2 (1:1000, Cell Signaling Technology, Boston, MA, USA), β-catenin (1:1000, Cell Signaling Technology, Boston, MA, USA), Bax (1:500, Bioss, Beijing, China), and caspase-3 (1:500, Bioss, Beijing, China) overnight at 4 °C. After washing with TBS, the membranes were incubated with IRDye^® 800CW goat anti-rabbit IgG secondary antibody (1:18,000, LI-COR Biosciences, Shanghai, China) for 1 h at room temperature. The blots were imaged using an Odyssey laser imaging system (LI-COR Biosciences, Lincoln, NE, USA) and quantified using ImageJ software version 1.8.0. The protein levels were normalized to β-actin (1:10,000, BioWorld, Nanjing, China).

Total protein samples were extracted from liver tissues and HepG2 cells, loaded on 12% sodium dodecyl sulfate-polyacrylamide gels (Beyotime, Biotechnology, Jiangsu, China), and then transferred to polyvinylidene difluoride membranes (BosterBiological Technology, Wuhan, China). The membranes were incubated with primary antibodies toNLRP3 (Cell Signaling Technology, Boston, MA, USA; #15101), caspase-1 (Cell Signaling Technology; #2225), interleukin (IL)-1β (Cell Signaling Technology; #12507), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology; #5174) (1:1000) at 4°C overnight, followed by goat anti-mouse IgG-HRP (Transgen, HS201-01, 1:5000) or anti-rabbit IgG-HRP (Transgen, HS101-1, 1:5000) for 1 h. GAPDH was used as an internal control. The protein bands were measured and analyzed using Quantity One software. Six replicates per group were used for each western blot and two to three independent experiments were performed.