Sc 56036

Proteins were extracted from the ileum as previously described [24 (link)]. The primary antibodies were as follows: Anti-claudin 1 (1:500 dilution, 13050-1-AP, Proteintech Group, Chicago, IL, USA), anti-occludin (1:1000 dilution, ab31721, Abcam, Cambridge, UK), anti-caspase-1 (1:200 dilution, sc-56036, Santa Cruz Biotechnology, Dallas, TX, USA), anti-caspase-4 (1:1000 dilution, GTX113639, GeneTex, San Antonio, TX, USA), anti-GSDMD (1:500 dilution, sc-81868, Santa Cruz Biotechnology) and anti-Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), (1:1000 dilution, 60004-1-Ig, Proteintech Group). Horseradish peroxidase-conjugated secondary antibodies used were goat anti-mouse IgG (1:5000 dilution, SA00001-1, Proteintech Group) or goat anti-rabbit IgG (1:5000 dilution, SA00001-2, Proteintech Group). The bands were visualized using a Tanon-5200 gel image system (Tanon, Shanghai, China). The intensity of bands was quantified by densitometric analysis using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

BEAS-2B cells were lysed in a buffer containing 20 mM Tris, 150 mM NaCl, 1% [vol/vol] Nonidet P-40, 1 mM DTT, 1% [vol/vol] Protease Inhibitor Cocktail, 1% [vol/vol] Phosphatase Inhibitor Cocktail. Total protein content was determined by the Bio-Rad DC Protein Assay kit (Bio-Rad, Hercules, CA), according to manufacturer’s instructions. 20 μg protein for WCL or 15 μL of concentrated lavage or cell culture supernatants was loaded onto polyacrylamide gels. After transfer of proteins to a nitrocellulose membrane, primary antibodies against thioredoxin (ab16965, Abcam -kindly gifted by Dr. Haendeler) and caspase-1 (sc-56036, Santa Cruz) were applied at a dilution of 1:1500 and 1:300 respectively, followed by HRP conjugated secondary antibodies. SuperSignal west femto maximum sensitivity ECL Substrate was used to visualize the proteins of interest (Thermo Scientific) and images were taken on the AIDA image analyzer.

Proteins were obtained by RIPA lysis buffer containing fresh protease and phosphate inhibitor mixture and boiled for denaturation. Protein concentration was determined by BCA protein assay. Cell lysate was then prepared for western blotting using a 10% polyacrylamide gel and 30 μg of protein. After electrical transfer and blocking with 5% BSA, the membrane was incubated with primary antibodies at 4°C overnight. Antibodies against β-actin (A5441) were obtained from Sigma–Aldrich; antibodies against TFEB (ab270604), NLRP3 (ab263899), PINK1 (ab23707), and Parkin (ab77924) were purchased from Abcam; and antibodies against GAPDH (SC-365062), caspase-1 (SC-56036), and P62 (SC-48402) were obtained from Santa Cruz Biotechnology.

NK (purity, >98%) was acquired from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China) (495–31–8) (250 mg administered). Lipopolysaccharide (LPS, 297–473–0) was acquired from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against α-SMA (bs-10196R) and collagen type I (collagen-I) (bs-0578R) were obtained from Bioss (Woburn, MA, USA). Antibodies against PPARα (sc-398394), SREBP-1 (sc-365513), caspase-1 (sc-56036), IL-23 (sc-271279), ASC (sc-514414), Lipin-1 (sc-376874), Nur77 (sc-365113), NLRP3 (ab4207), P2X7r (sc-514962), and IL-1R1 (sc-393998) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against NLRP3 (ab4207), Lipin-1 (ab181389), P2X7r (ab307718), MPO (ab208670), and GAPDH (ab8245) were obtained from Abcam (Cambridge, MA, USA). Csn-B (a Nur77 agonist, 321661–62–5) was purchased from Beyotime Biotechnology (Shanghai, CN, USA). rNur77 (ab152448) was purchased from Abcam (Cambridge, MA, USA).

Protein lysates were added to lyse mouse heart tissues and myocardial cells. The supernatant in different groups was gathered after centrifugation (12,000 rpm for 15 min). The proteins were split by SDS–PAGE before they were devolved into PVDF membranes. After the transfer, we blockaded the membrane with 5% skim milk for 1 h at 37 °C. The primary antibodies against NLRP3 (NBP2-12446, 1:1000; Novus Biologicals, Littleton, CO, USA), caspase-1 (sc-56036, 1:200; Santa Cruz Biotechnology, Dallas, TX, USA), and GSDMD (sc-393581, 1:200; Santa Cruz Biotechnology, Dallas, TX, USA) were washed with TBST solution and blocked overnight at 4 °C. The excess antibody was washed thoroughly with TBST solution 3 times for 10 min. The immunoreactive bands of the secondary antibody to goat anti-rabbit (A0208, 1:2000; Beyotime Biotechnology) and goat anti-mouse (A0216, 1:2000; Beyotime Biotechnology) with the corresponding bound secondary antibody were then visualized with ECL chemiluminescence and quantified by image software. GAPDH, a reference quantity, was used to qualify the protein expression.

For immunohistochemistry, eyes and adnexae from each group/time point (n = 6/group) were excised, embedded in optimal cutting temperature compound (VWR, Suwanee, GA, USA), and flash frozen in liquid nitrogen. Sagittal 8 µm tissue sections were cut with a cryostat (HM 500; Micron, Waldorf, Germany) and placed on glass slides that were stored at −80 °C.
Immunofluorescent staining was performed in frozen tissue sections with rat monoclonal antibody anti-NLRP3 (MAB7578, 10 µg/mL, R&D Systems, Minneapolis, MN, USA), anti-ASC (SC-22514-R, 1 µg/mL, Santa Cruz Biotechnology, Dallas, TX, USA), anti-caspase-1 (SC-56036, 1 µg/mL, Santa Cruz Biotechnology, Dallas, TX, USA), and goat anti-IL-1β (1:50 dilution, #12426, Cell Signaling Technology, Beverly, MA, USA). Secondary goat anti-rabbit or donkey anti-goat Alexa Fluor 488-conjugated antibodies were used, as previously described [30 (link)]. The images were captured and photographed by a laser scanning confocal microscope (LSM 510, with Kr–Ar and He-Ne laser; Carl Zeiss Meditec, Inc., Thornwood, NY, USA).