Enhanced chemi luminescence (ecl)

Proteins were extracted from H2170 cells using RIPA solution (Invitrogen). After quantification using BCA assay (Invitrogen), the same amount of proteins were denatured at 95 °C for 10 min, separated by electrophoresis on SDS-PAGE gels, and transferred onto PVDF membranes. The membranes were incubated with 5% fat-free milk (PBS) at room temperature for 2 h before incubation with primary antibodies against cyclin D1 (ab194972, Abcam) and GAPDH (ab9845, Abcam) for 16 h at 4 °C. After that, the membranes were incubated with HRP IgG secondary antibody (ab6721, Abcam) for 3 h at 25 °C. Protein signals were developed using ECL (Invitrogen), detected, analyzed, and normalized to the control using Quantity One software.

Brain tissues or treated cells were lysed with RIPA buffer supplemented with phosphatase and protease inhibitors and were then collected for protein extraction. The protein concentration was determined using a BCA kit. An equal amount of protein samples was loaded on sodium dodecyl sulfate (SDS)-polyacrylamide gel for electrophoresis at 120 V for 90 min. Subsequently, proteins were transblotted onto polyvinylidene fluoride (PVDF) membranes by wet transfer (400 mA, 90 min). The membrane was blocked by 5% non-fat milk in Tris-buffered saline with Tween-20 (TBST) for 2 h at room temperature and then incubated overnight at 4 °C with the primary antibodies (ChemR23, PA5-50932, 1:500, Invitrogen, USA; NLRP3, GB114320, 1:1000, Servicebio, China; apoptosis-associated speck-like protein containing a CARD (ASC), 10500-1-AP, 1:1000, Proteintech, IL, USA; Caspase-1 p20, AG-20B-0042, 1:1000, AdipoGen, Switzerland; GSDMD-N terminal (GSDMD-N), GB114198, 1:1000, Servicebio, China; tubluin, 11224-1-AP, 1:1000, Proteintech, IL, USA). After washing with TBST, the membranes were incubated with the secondary antibody (anti-rabbit and mouse IgG, 1: 5000; Santa Cruz Biotechnology, USA) for 2 h at room temperature, and then reacted with enhanced chemiluminescence (ECL, Invitrogen, USA). The result of chemiluminescence was recorded and analyzed with ImageJ software.

Cells were lysed using RIPA buffer with 1% phenylmethanesulfonyl fluoride (Beyotime Biotechnology, Jiangsu, China). Protein concentration was measured using the BCA Protein Assay Kit (Invitrogen, Waltham, MA, USA). Protein (20 µg) was separated on 12% SDS-PAGE, and then transferred to PVDF membranes (Merck Millipore, Tullagreen, Carrigtwohill, Co. Cork, Ireland). Membranes were blocked with 5% skim milk for 1 h and washed three times with TBST. The membranes were incubated overnight with primary anti-HBx (#ab39716; 1:1000; Abcam, Cambridge, MA, USA) and anti-β-actin antibodies (#BM0627; 1:20000; Boster, Wuhan, China) at 4 °C. Then, membranes were washed and incubated with HRP-conjugated secondary antibody at room temperature for 1 h. Proteins were visualized by ECL (Invitrogen, Waltham, MA, USA).