Sc 514

By using ice-cold RIPA buffer the total protein was isolated from hippocampal tissues. BCA Protein Assay Kit (Thermo Fisher Scientific, USA) was used to measure the protein concentrations. A nitrocellulose membrane (Millipore Co., USA) was used for immuno-blotting where the protein samples (30 μg) were separated using SDS–polyacrylamide gel electrophoresis and then transferred to. For 1 h the membrane was blocked with 5% skim milk in Trisbuffered saline (150 mM NaCl, 0.1% Tween 20, 20 mM Tris, pH 7.4). Then the proteins were detected by incubation with primary antibodies against HMGB1, RAGE at 4 °C overnight. Later the membrane was washed for 3 times in Tween 20 + PBS and was incubated by the horseradish peroxidase-conjugated secondary antibodies for 2 h. The immuno-blots were visualized by a Millipore ECL Western Blotting Detection System and the variables protein expression levels were normalized to those of β-actin.
The following antibodies were used: anti-β actin (1:2,000, sc-130657, Santa Cruz Biotechnology), anti-HMGB1 (1:1,000, ab18256; Abcam), anti-RAGE antibody (1:2,000, sc8230; Santa Cruz), anti-NLRP3 antibody (1:2,000, sc-66846, Santa Cruz Biotechnology), anti-ASC antibody (1:2,000, sc-22514-R, Santa Cruz Biotechnology), pro and cleaved Caspase1 antibody (1:2,000, sc-514, Santa Cruz Biotechnology) and pro and mature IL-1β (1:1,000, AF-401-NA, R&D Systems).

Polysulfide donor Na₂S₄ was purchased from Dojindo Molecular Technologies Dojindo (Kumamoto, Japan). Cisplatin and NaHS were bought from Sigma-Aldrich (St. Louis, MO, USA). GYY4137 was synthesized as we previously depicted [72 (link)]. A selective and reversible inhibitor of IKKβ (IKK-2), SC-514, was procured from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). A selective STAT3 inhibitor HO-3867 was obtained from Medchem express (Princeton, NJ, USA). Antibodies against IL-6, COX-2, F4/80, Biotin, Lamin B1, and β-actin, and the secondary antibodies, were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Antibodies against TNF-α, IL-1β, phosphorylated and total IκBα, NF-κB, phosphorylated and total STAT3, and phosphorylated and total IKKβ were obtained from Abcam (Cambridge, MA, USA). Dulbecco’s modified Eagle’s medium (DMEM), streptomycin/penicillin, trypsin, and fetal bovine serum (FBS) were provided by Hyclone Laboratories (South Logan, UT, USA). The radio-immunoprecipitation assay (RIPA) lysis buffer was purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Goat anti-mouse IgG (H+L) Highly Cross-Adsorbed Alexa Fluor 594 Secondary Antibody was procured from Invitrogen (Carlsbad, CA, USA). The specific primers used in this study were synthesized by Integrated DNA Technologies (Singapore).

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.

Proteins were obtained from supernatants of inflammasome-stimulated BMMs by trichloroacetic acid (TCA) precipitation (10% TCA for 10 min on ice). Pellets were dissolved in 2x Laemmli buffer and pH was readjusted with 1 M NaOH. Samples were then loaded onto 12% gels followed by SDS-PAGE and transferred to 0.2 μm PVDF membranes. The level of IL-1β and caspase-1 in culture supernatants was measured by immunoblotting with anti-IL-1β (AF-401-NA; R&D System; Minneapolis, MN) or anti-murine-caspase1 (SC-514; Santa Cruz; Santa Cruz, CA).

Protein (20–50 μg) was extracted from the ipsilateral spinal cord dorsal horn and separated with 10% SDS-PAGE gel, transferred onto a nitrocellulose membrane, and incubated with antibody against CXCR4 (1:1000; ab124824, Abcam), VDUP-1 (1:1000; sc-271238, Santa Cruz Biotechnology), NLRP3 (1:500; NBP1-77080, Novus Biologicals), apoptosis-associated speck-like molecule containing CARD domain (ASC; 1:1000; sc-22514-R, Santa Cruz Biotechnology), Caspase1 (1:1000; ab-1872, Abcam), Caspase1 p10 (1:1000; sc-514, Santa Cruz Biotechnology), and IL-1β (1:1000; ab9722, Abcam) or control β-Actin (1:5000; YM3028, ImmunoWay). Blots were washed and incubated in HRP-linked anti-rabbit IgG antibody (1:5000; 7074, Cell Signaling Technology) and HRP-linked anti-mouse IgG antibody (1:5000; 7076, Cell Signaling Technology). Protein blots were visualized using Clarity ECL Substrates (Biorad).

We detected IL‐1β and caspase‐1 cleavage by immunoblotting. Isolated protein from supernatant was precipitated with methanol and chloroform. Briefly, 500 μL supernatant is fully mixed with 500 μL of methanol and 125 μL of chloroform. After centrifuge, a protein layer would be visible. Washed them with 500 ul methanol and dried. Then, boiled protein sample in the 40 ul loading buffer at 95°C for 10 minutes and separated by SDS/PAGE, followed by transfer to polyvinylidene difluoride membranes (PVDF). Immunoblots were incubated with primary antibodies against caspase‐1 (sc‐514, Santa Cruz) and IL‐1β (5129‐100, BioVision). Immunoblots from cell lysates incubated with antibodies against NLRP3 (15 101, Cell Signaling Technology) and NF‐κB (pp65). Proteins were detected by enhanced chemilumiescense.