Af 401 na

Neutrophil lysates (20–30 μg protein) were fractionated in 12% SDS–PAGE, transferred onto nitrocellulose membranes, and incubated with primary antibodies—mouse IL-1β (R&D Systems, AF-401-NA at 1:1,300), mouse Caspase-1 p10 (Santa Cruz, SC-514 at 1:200), or with antibodies targeted to the intracellular C terminus or extracellular region of P2X₇R (Alomone Labs, catalog# APR 004 for the C terminal, and APR 008 for the ecto-domain, both at 1:200 dilution). These antibodies recognize mouse and human receptors. Loading controls were shown using antibodies to β actin (Sigma Aldrich A3854, 1:50 000). Reactivity was determined using HRP-conjugated secondary antibodies (Santa Cruz) and developed using Supersignal West Femto Maximum Sensitivity Substrate (Pierce). Images were cropped for presentation; full size images are presented in Supplementary Fig. 5.

Antibodies against NLRP3 (Adipogen, Cryo2) (Sigma, HPA012878), IL-1β (R&D systems, AF-401-NA), caspase-1 (Santa Cruz, SC-154), MAP1 (Sigma, HM-1), NEK7 (Abcam, ab133514), MAP4 (BD, 611026), α-tubulin (MBL, PM054), acetylated tubulin (Santa Cruz, SC-23950), γ-tubulin (Sigma, T5326), Tom20 (Santa Cruz, FL-145, SC-11451), ERK2 (Santa Cruz, C-14), HA tag (Santa Cruz, y-11), Flag tag (Sigma, F1804) were used for western blots. Antibodies against MARK4 (Cell Signaling, 4834) (Abcam, ab124267) (house-made by MRC-PPU, the University of Dundee, UK), NLRP3 (Sigma, HPA012878; Abcam, ab4207), ASC (Enzo, ADI-905-173) (Santa Cruz, SC-33958) were used for in situ proximity-ligation assay. Antibodies against markers CD11b (BIOLEGEND, 101217, 1 in 800 dilution), F4/80 (ebiosciences, 25-4801-82, 1 in 200 dilution), Ly6G (BD, 551461, 1 in 400 dilution), and Ly6C (AbD SeroTec, mca771a647, 1 in 400 dilution) were used for flow cytometry. See Supplementary Fig. 15d for gating strategy for peritoneal exudates.

Cell lysates and supernatants (reduced and denatured) were separated on 4–12% gradient gels (Invitrogen) and protein transferred nitrocellulose membrane (Amersham) for detection. Membranes were blocked with 5% skimmed milk in PBS containing 0.1% Tween 20 (PBST) for 1–2 h and were then probed overnight with primary antibodies (all diluted 1/1,000 unless noted otherwise) to mouse β-actin (Sigma; A-1978), cIAP1 (1/500 dilution, ALX-803-335;Alexis Bio-chemicals), RIPK3 (Axxora; PSC-2283-c100), RIPK1 (BD Transduction Laboratories; 610458), MLKL (in-house; 3H1), pro and mature IL-1β (R&D Systems; AF-401-NA), pro- and cleaved-caspase-1 (Santa Cruz; sc-514), Adipogen (AG-20B-0042-C100), pro-caspase-8 (in-house), cleaved caspase-8 Asp387 (9429; Cell Signaling) and Ubiquitin (3933; Cell Signaling). Relevant horseradish peroxidase-conjugated secondary antibodies were applied for 1–2 h. Membranes were washed four to six times in PBST between antibody incubations and all antibodies were diluted in PBST containing 5% skimmed milk. Membranes were developed using ECL (Millipore). For images cropped for presentation, the full-size images are presented in Supplementary Figs 9–14.