Pyrin

Total proteins were separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against IL-1β (#AF-401-NA, R&D system, Minneapolis, MN, USA), precursor and p10 subunit of caspase-1 (#sc-514, Santa Cruz, Dallas, TX, USA), NLRP3 (#AG-20B-0014, AdipoGen, San Diego, CA, USA), HMGB-1 (#AB79823, Abcam, Cambridge, MA, USA), P2X₇ (#sc-514962, Santa Cruz, Dallas, TX, USA), pyrin (#sc-390938, Santa Cruz, Dallas, TX, USA) and α-tubulin (#T5168, Sigma-Aldrich, St. Louis, Missouri, USA). The expression of low-molecular-weight (LMW) surface layer proteins in the cell culture supernatant was also detected using rabbit anti-LMW SLP BAA 1805 serum (customized by Abnova, Taipei, Taiwan).

Brain protein lysates were obtained, as described in [62 (link),63 (link)], and 25 μg of protein lysates was then resolved via immunoblotting for the expression of inflammasome signaling proteins, as described in [64 (link)]. Primary antibodies used in this study were against the following proteins: NLRP1 (Novus Biologicals, Centennial, CO, USA), NLRP3 (Novus Biologicals Centennial, CO, USA), AIM2 (eBioscience, San Diego, CA, USA), NLRC4 (Novus Biologicals, Centennial, CO, USA), pyrin (Santa Cruz Biotechnology, Inc. Dallas, TX, USA), caspase-1 (Novus Biologicals, Centennial, CO, USA), ASC (Santa Cruz), IL-1β (Cell Signaling, Technology, Inc. Danvers, MA, USA), caspase-8 (Novus Biologicals, Centennial, CO, USA), caspase-11 (Novus Biologicals, Centennial, CO, USA), CD63 (Novus Biologicals), β-actin (Sigma-Aldrich, St. Louis, MO, USA) and GAPDH (Sigma Aldrich, St. Louis, MO, USA).

Brain and heart protein lysates were obtained as described in (Mejias et al., 2018 (link)), and lysates were then resolved by immunoblotting for the expression of inflammasome signaling proteins as in (Cyr and de Rivero Vaccari, 2023a (link)). Briefly, lysates were resolved in 4–20% Criterion TGX Stain-Free precast gels (Bio-Rad), using antibodies at a dilution of 1: 1,000. Primary antibodies used in this study were against the following proteins: NLRP1 (Novus Biologicals), NLRP3 (Novus Biologicals), AIM2 (eBioscience), Pyrin (Santa Cruz), caspase-1 (Novus Biologicals), ASC (Santa Cruz), IL-1β (Cell Signaling), caspase-8 (Novus Biologicals), CD63 (Novus Biologicals), and β-actin (Sigma Aldric). Quantification of band densities was done using the UN-SCAN-IT gel 6.3 Software (Silk Scientific Corporation). Membranes were imaged using the ChemiDoc Touch Imaging System (BioRad) following chemiluminescence.