Anti caspase 1 antibody

Magnolol was purchased from National Institutes for Food and Drug Control (Beijing, China). Ethanol was bought from Beijing Chemical Works (Beijing, China). AST, ALT, SOD, iNOS, and GSH-Px kit were provided by Nanjing Jiancheng Bio-engineering Institute (Nanjing, China). Cox-2 and CYP2E1 Elisa kits were bought from Shanghai Lanpai Biotechnology co. LTD. Antibodies against GAPDH, p-AKT, AKT, Nrf2, HO-1, NLRP3, p-PI3K, and PI3K were purchased from Boster bioengineering co. LTD (Wuhan, China). Antibodies against PPARγ and Caspase-3 were obtained from Cell Signal Technology (Boston, MA, USA). Anti-Caspase-1 antibody was bought from Abcam (Cambridge, MA, USA). Additionally, all other chemicals were provided by Beijing Chemical Works (Beijing, China), if not otherwise indicated.

LPS (Escherichia coli serotype O127:B8) was from Sigma Aldrich (Allentown, PA, USA). ATP disodium salt and phorbol 12-myristate 13-acetate (PMA) were from MedChemExpress (Monmouth Junction, NJ, USA). A stock solution of LXA₄ (Cayman Chemical, Ann Arbor, MI, USA) was stored at −80 °C until being diluted in sterile 0.9% saline or cell culture medium immediately before use. Anti-NF-κB p65, -ALOX12, -ALOX15, -ALOX15B, -AT1R, -β-actin, and -Lamin B antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti phospho-P38 and P38 antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-Caspase-1 antibody was from Abcam Company (Cambridge, UK). Anti-CD68 antibody was from Proteintech Group, Inc. (Wuhan, China). APC-conjugated Rat Anti-Mouse CD19 and PE-conjugated Rat Anti-Mouse CD5 were from BD Biosciences (San Diego, CA, USA). Mouse ALOX5 monoclonal antibody (BD Biosciences) was used for western blotting (WB), whereas rabbit ALOX5 polyclonal antibody (Cayman Chemical) was used for immunohistochemistry (IHC). RIPA Lysis Buffer and Nuclear and Cytoplasmic Protein Extraction Kit were from Beyotime Institute of Biotechnology (Shanghai, China). BCA protein assay kit was from Pierce (Rockford, IL, USA).

Total proteins were extracted from lysates of THP-1 cells treated with plasma from active AOSD patients or healthy controls. The samples were run on 10% SDS-PAGE and then transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). The blots were blocked with 5% milk in PBS with 0.1% Tween-20 (PBST) (Bionovas, Inc., Washington, DC, USA) for 30 min at room temperature, and subsequently incubated with specific anti-CLEC5A antibody (Aviva Systems Biology, San Diego, CA, USA), anti-NLRP3 antibody (Cell Signaling Technology, Beverly, MA, USA), anti-caspase-1 antibody (Abcam, Cambridge, MA, USA), anti-IL-1β antibody (Novus Biologicals, LLC, Littleton, CO, USA), anti-IL-18 antibody (Medical & Biology Laboratories Co, Ltd., Naka-ku, Nagoya, Japan), and anti-α-tubulin (1: 5000, Santa Cruz Biotechnology, Dallas, Texas, USA) at 4°C overnight. After washing with PBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (Thermo Fisher Scientific Inc., Pittsburgh, PA, USA). Immunoreactive bands were incubated with an ECL detection system (Advansta, Menlo Park, CA, USA) and visualized by radiographic film. The band intensity was quantitated by ImageJ software as described previously [22 (link)]. The protein levels of NLRP3, caspase-1, IL-1β, and IL-18 were normalized to α-tubulin.

MLEC lysates were separated by 5 and 10% SDS-polyacrylamide gel electrophoresis, and then were transferred onto polyvinylidene difluoride membranes. The membranes were blocked with blocking buffer (LI-COR Biosciences, Lincoln, NE) for 1 h at room temperature and incubated with the anti-caspase-1 antibody (Abcam, Cambridge, MA) at 4 °C overnight. After washing, membranes were incubated with secondary antibodies (LI-COR Biosciences) for 1 h. Protein bands were visualized using the Odyssey System (LI-COR Biosciences).