Caspase 11

Protein levels of inflammasome signaling proteins were determined in the cytosolic and mitochondrial fractions as described in [7 (link)]. Briefly, protein lysates were resolved in 10–20% Criterion TGX Stain-Free precasted gels (Bio-Rad), using antibodies (1:1000 dilution) to NLRC4 (Novus Biologicals, cat# NBP2–41124), caspase-1 (Novus Biologicals, cat# NB100–56565), caspase-11 (Novus Biologicals, cat# MAB8648), ASC (Santa Cruz, cat# sc-271054), IL-1β (Cell Signaling, cat# 12242S), IL-18 (Abcam, cat# ab71495), HSP60 (Cell Signaling, cat#12165) and β-actin (Sigma Aldric, cat# A5441). Quantification of band densities was done using the UN-SCAN-IT gel 6.3 Software (Silk Scientific Corporation). Chemilluminescence substrate (LumiGlo, Cell Signaling) was imaged with the ChemiDoc Touch Imaging System (BioRad).

Total of mouse splenocytes, CD11b⁺ cells, BMDM, or monocytes from patients were lysed with RIPA buffer solution with a protease inhibitor cocktail. After 15 min on ice, lysates were centrifuged at 13,000 × g for 10 min at 4 °C. The proteins were separated in a 12%-acrylamide (caspase-11 and caspase-8) or 4–12% acrylamide (caspase-1/caspase-4/GSDM-D) NUPAGE Bis-tris Protein gels (Invitrogen) and transferred onto nitrocellulose membranes. The membranes were incubated with caspase-11 (Novus Biologicals, 1:1000), caspase-1 (Adipogen, 1:1000), caspase-4 (Cell Signaling, 1:1000), anti-mouse and anti-human caspase-8 (Enzo Life Science, 1:1000), anti-mouse cleaved caspase-8 (Cell Signaling, 1:500), anti-GSDM-D (sigma, 1:1000) and β-actin (Sigma, 1:2000) specific antibodies, then incubated with HRP-conjugated secondary antibodies (Jackson ImmunoResearch, 1:25,000) and detected with Clarity Max ECL Substrate (Biorad) using ImageLab Touch Software V6.0.1 (Bio-Rad). Bands quantification was performed with ImageStudio V5.2. Uncropped western blot is available in the Supplementary Information file (Supplementary Figs. 5–11).

Cells were lysed in ice-cold PBS containing a cocktail of protease and phosphatase inhibitors to isolate proteins. Protein concentration was determined according to the Bradford method [59 (link)] using γ-globulin as a standard.
Aliquots of 15 μg of protein were separated on 10% or 15% acrylamide gel by sodium-dodecyl sulfate polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and incubated overnight at 4 °C with specific primary antibodies against mPGES-1, IL-18 (Abcam^®, Cambridge, UK), COX-2, iNOS, pJNK, JNK, P38, pP38, pERK1/2, ERK ½, Nrf-2, pSTAT-3, pJAK2, NLRP3, ASC, H3K18ac, H3K27me3, H3K9me3, H3 (Cell Signaling^®, Danvers, MA, USA), HO-1 (Henzo^®, Madrid, Spain), caspase-1 and caspase-11 (Novus Biologicals^®, Littleton, CO, USA). Nitrocellulose membranes were incubated for 2 h in a blocking solution with antirabbit horseradish peroxidase-labeled (HRP) secondary antibody (Cell Signaling^®, Danvers, MA, USA) or antimouse HRP secondary antibody (Dako^®, Atlanta, GA, USA). β-Actin primary antibody (Abcam^®, Cambridge, UK) was employed to demonstrate equal loading. The immunosignals were captured using Amersham Imager 600 from GE Healthcare^® (Buckinghamshire, UK) and analyzed and quantified by Image Processing and Analysis in Java (ImageJ^®, Softonic) and expressed comparing with the DMSO-LPS treated cells.