Anti p65

Animals were perfused with 4% paraformaldehyde, tissue was embedded in optimal cutting medium, and 40 μm frozen sections were prepared. For fluoromyelin staining, slides were rehydrated in PBS for 20 min and then flooded with 1:300 diluted stain for 20 min. Counterstaining was done using Hoechst stain for 5 min, and slides were mounted using vectashield mounting medium (Vector Laboratories). Slides were imaged using the Zeiss LSM 700. For antibody staining, sections were incubated with primary anti-RelB, anti-GFAP, anti-p65, anti-phospho-p65 (all 1:300, Cell Signaling), anti-CC1 (1:200, Millipore), or anti-Iba1 (1:500, Wako) antibodies overnight at 4 °C. Subsequently, sections were incubated with Alexa Fluor 488 or Alexa Fluor 594 secondary antibodies (1:500, Invitrogen) for 1 h at room temperature. Slides were mounted and imaged as described above. No fluorescence crossover was found between the channels, and images were collected separately using the appropriate laser excitation. Images were analyzed using ImageJ.

Protein extracts (50 μg) were run on 10% SDS‐PAGE. The protein was then transferred to a polyvinylidene difluoride membrane (PVDF, Amersham Biosciences). The membrane was blocked for 1‐hour at room temperature with 10% BSA in phosphate‐buffered saline/0.05% Tween 20. The blots were incubated overnight at 4°C with anti‐α‐SMA, anti‐collagen IV, anti‐fibronectin, anti‐phosp‐IκB, anti‐IκB, anti‐NRF, anti‐p65, anti‐p50, anti‐Histone H1 or anti‐Tubulin antibody and secondary antibody (Cell Signalling, Danvers, MA). The protein expression was visualized using enhanced chemiluminescence reagents (Bio‐Rad, Hercules, CA). The amounts of the proteins were analysed using Image J analysis software.

The whole cells were washed and lysed in RIPA buffer (89900, Thermo Scientific) supplemented with PMSF (phenylmethylsulfonyl fluoride) and protease inhibitors. Protein concentrations were determined with a BCA assay kit (Pierce, Rockford, IL, USA). Then the lysates were mixed with loading buffer, analyzed by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA). After blocked with 5% skim milk in TBST at room temperature for 1 h, the membranes were incubated with different primary antibodies, including anti-DDX54 (1:1000, Proteintech, China), anti-P65, anti-p-P65, anti-AKT, anti-p-AKT, anti-mTOR, anti-p-mTOR, (1:1000, Cell Signaling Technology, Danvers, MA, USA), and anti-GAPDH (1:5000, Yeason, China) in 5% milk/TBST buffer at 4°C overnight, and then probed with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG (1:5000, Jackson Immunoresearch Laboratories, West Grove, PA, USA) for 1 h. After washing three times with TBST, the membrane was developed with enhanced chemiluminescent plus substrate (Merck Millipore, Billerica, MA, USA), and the signal was recorded by Fluorchem E System (ProteinSimple, Santa Clara, CA, USA).

Dulbecco’s modified Eagle medium and penicillin–streptomycin solution were purchased from GIBCO (Grand Island, NY, USA). Fetal bovine serum was provided by Hyclone (Logan, UT, USA). Monoclonal rabbit antibodies, including anti-p38 MAPK, anti-phospho-p38 MAPK, anti-ERK1/2, anti-phospho-ERK1/2, anti-JNK, anti-phospho-JNK, anti-p65, anti-phospho-p65, NLRP3, and cleaved caspase-1 were obtained from Cell Signaling Technology (Boston, MA, USA). Receptor for AGE (RAGE) monoclonal rabbit antibody was purchased from AbCam (Cambridge, MA, USA). HRP-marked anti-β-actin antibody was supplied by Biorad (San Diego, CA, USA). 4′-Methoxyresveratrol (3,5-dyhydroxy-4′-methoxylstilbene) was purchased from Great Forest Biomedical (Hangzhou, China). BSA was obtained from ABCONE (Shanghai, China). KI and acetic acid were obtained from Sangon Biotech (Shanghai, China). Methylglyoxal, 2’,7’-dichlorodihydrofluorescein diacetate and other reagents were purchased from Sigma (St. Louis, MO, USA).

Total protein extracts or cytoplasmic and nuclear fractions were analyzed by SDS-PAGE and transferred onto a PVDF membrane (Roche Applied Science, Switzerland). Blots were blocked with PBS containing 5% non-fat dry milk and 0.1% Tween and then incubated with primary antibody. Primary antibodies were mouse anti-flag and anti-V5 antibodies (Invitrogen, USA); mouse anti-actin, rabbit anti-hepsin, and normal IgG antibodies (Santa Cruz Inc, USA); anti-phospho-p38, anti-phospho-C/EBP-β, anti-phospho-ERK, anti-phospho-JNK, anti-p38 anti-C/EBP-β, anti-ERK, anti-JNK, anti-p65, anti-p50, anti-histone H3 and anti-α-tublin (Cell Signal Technology, USA); anti-C3, anti-HBc and anti-HBx antibody (Abcam, UK).

Carnosic acid, 5-aminosalicylic acid (5-ASA), sodium carboxymethyl cellulose, haematoxylin and eosin were purchased from Sigma Aldrich (St. Louis, MO). Dextran sodium sulfate was purchased from MP Biomedicals (Solon, OH). Anti-p65 (#8242), anti-phospho-p65 (#3039), anti-IĸBα (#9242), anti-phospho-IĸBα(#2859), anti-Stat3 (#4904), anti-phospho-Stat3 (#9145), anti-JNK (#9252), anti-phospho-JNK (#4668), anti-c-Jun (#2315), anti-phospho-c-Jun (#2361), anti-NLRP3 (#15101), anti-ASC (#67824), anti-Ubiquitin (#3933), iNOS (#13120), anti-H3k27Me3 (#9733), anti-H3k4Me3 (#9751) and anti-β-actin (#4970) antibodies were purchased from Cell Signaling Technology (Beverly, MA). Anti-Keap1 (ab150654), anti-Nrf2 (ab62352) and Cullin3 (ab75851) antibodies were purchased from ABCAM. Anti-caspase1 (sc-56036) antibody was purchased from Santa Cruz Biotechnology, TRIzol reagent, TaqMan primers and TaqMan PCR Master Mix were purchased from Invitrogen (Carlsbad, CA).