Chemiluminescence substance

Hepatocytes were washed with cold PBS, collected in cell lysis buffer, sonicated, and centrifuged (10,000g for 15 min), and the protein-rich supernatant was transferred to a new tube. Protein concentrations were calculated using the BCA protein assay kit (Pierce Biotechnology, Rockford, IL). Samples were then mixed with loading buffer and run on an SDS-polyacrylamide gel. This gel was then transferred to a cellulose membrane. The membrane was blocked in 5% milk in TBS-Tween 20 for 1 hour and then incubated in primary antibodies. Antibodies utilized were iNOS, NRF2 (Abcam, Cambridge, MA), LC3 (Novus Biologicals, Littleton, CO, USA), and β-actin (Abcam). Membranes were then washed in TBS-Tween 20 for 30 minutes, placed in secondary antibody for 1 hour, and then washed for 1 hour in TBS-Tween 20 prior to being developed using chemiluminescence substance (Thermo Fisher, Rockford, IL, USA).

Cells were firstly lysed in RIPA buffer (Sigma-Aldrich, USA) and protein concentrations of each homogenate were measured using a commercial BCA protein assay Kit (Thermo Fisher Scientific, USA). 30 μg of proteins were separated on SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was then blocked in 5% skimmed milk in TBST, and stained overnight at 4 °C with primary antibodies against Nrf2 (1:1000, ab137550, Abcam), HO-1 (1:2000, ab13243, Abcam), eNOS (1:5000, ab76198, Abcam), NQO1 (1:10,000, ab80588, Abcam), caspase-1 (1:1000, ab238979, Abcam), NLRP3 (1:1000, ab263899, Abcam), ASC (1:1000, ab180799, Abcam), HMGB1 (1:10,000, ab79823, Abcam), or β-actin (1:5000, ab115777, Abcam). Samples were then stained with goat anti-rabbit or goat anti-mouse IgG secondary antibody (1:10,000, ab205718/ab205719, Abcam) at room temperature for 2 h. Bands were developed using chemiluminescence substance (Thermo Fisher Scientific, USA). The proteins were quantified using Quantity One software (Bio-Rad, USA).

Primary mouse hepatocytes were washed with cold PBS, collected in lysis buffer (Cell Signaling Technology, Danvers, MA), sonicated, and centrifuged (13,000 rpm for 15 min), and then the supernatant was collected. Protein concentrations were determined using the bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Rockford, IL). Loading buffer was added to the samples and separated by 10% and 15% sodium dodecyl sulfate-polyacrylamide (SDS PAGE) gel. The gel was then transferred to a polyvinylidene fluoride membrane at 250 mA for 2 h. The membrane was blocked in 5% milk for 1 h and then incubated in primary antibody with 1% milk overnight. Membranes were washed in TBS-T (Tween) for 10 min and then placed in horseradish peroxidase secondary antibody for 1 h and washed for 1 h in TBS-T before being developed using a chemiluminescence substance (Thermo Fisher Scientific, Rockford, IL). The primary antibodies used were LC3B, SQSTM1/p62, Beclin-1 (Cell Signaling Technologies, Danvers, MA), LC3B (Novus Biologicals, Littleton, CO), Nrf2 (Santa Cruz Biotechnologies, Dallas, Texas), β-actin (Bio-vision, Milpitas, CA), and Myd88 and TIRAP (eBioscience, San Diego, CA). An ubiquitin enrichment kit from Pierce (Thermo Fisher Scientific, Rockford, IL) was used for ubiquitin immunoprecipitation and we followed the manufacturer's protocol.

Cells were lysed using the RIPA buffer and protein concentration was tested using BCA protein assay kit. The proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel electrophoresis and then transferred onto a nitrocellulose membrane (Milipore, Billerica, MA, USA). After blocked using BSA, the membranes were incubated with primary antibodies against RohA, ROCK1, ROCK2, and GAPDH (Cambridge, MA, USA). The bands were indicated with goat anti-rabbit IgG-HRP secondary antibody (1:2,000; Abcam Cambridge, MA, USA) and were exposed using chemiluminescence substance (Thermo Fisher Scientific, Waltham, MA, USA).