Anti caspase 4

Antibodies used for immunoblotting analysis were the following: anti-IL-1β (#12703), anti-caspase-1 (#3866), anti-caspase-4 (#4450), anti-caspase-5 (#46680), anti-MLKL (#14993), anti-phospho-MLKL (#91689), anti-RIP-3 (#13526), anti-phospho-RIP3 (#93654) and anti-Myc-tag (#2278) (Cell Signaling Technology); and anti-Flag (Sigma-Aldrich, F1804), anti-Bcl-2 (Santa Cruz Biotechnology, sc-783), anti-actin conjugated to horseradish peroxidase (Sigma-Aldrich, A3854), anti-GAPDH conjugated to horseradish peroxidase (Proteintech, HRP-60004), goat anti-rabbit HRP-linked antibody (#7074), and horse anti-mouse HRP-linked antibody (#7076) (Cell Signaling Technology). Anti-GSDMD was from (Novusbio, NBP2-33422). The LPS, nigericin and Z-VAD-FMK caspase inhibitor were purchased from Sigma-Aldrich. SYTOX Green nucleic acid stain was purchased from Invitrogen. Smac mimetic was from Tocris. Caspase-1, caspase-3 and Bcl-2 recombinant proteins were from EMD Millipore. Human TNFα was from R&D System. Smac mimic was from APExBIO.

THP‐1 cells were differentiated for 3 days in a culture medium supplemented with 0.1 μM of phorbol 12‐myristate 13‐acetate (PMA). Cells seeded in 12‐well plates were primed for 5–6 h with the indicated ligand. Primed cells were transfected with 2.5 μg LPS by using 0.25% v/v Fugene HD (Promega, Madison, WI, USA). Cells were washed twice, fixed in 4% paraformaldehyde, and permeabilized with 0.1% Triton X‐100. The cells were incubated with anti‐Myc (Genescript; diluted 1:500), FLAG (Cell Signaling; 1:1,000), GPx8 (GeneTex; 1:50), CD68 (Biolegend, USA; 1:100), CD163 (Leica Biosystems, Buffalo Grove, IL, USA; 1:200), CD103 (Leica Biosystems; 1:100), F4/80 (Bio‐Rad; 1:50), and anti‐caspase‐4 (Cell Signaling; 1:100) and then with either Alexa 488‐labeled or Alexa 647‐labeled antibody to rabbit or mouse IgG (Invitrogen; 1:300). Nuclei were stained with DAPI (4ʹ,6‐diamidino‐2‐phenylindole, Dojindo, Rockville, MD, USA). Co‐localization was quantified by Pearson's correlation coefficient by ImageJ software, in order to measure the strength of co‐localization between proteins; the formulas return a value between −1 and 1, where 1 indicates a strong positive relationship.