Sc 22514

THP-1 and HEK293T cells were lysed with RIPA buffer supplemented with cOmplete protease inhibitors (11697498001; Roche Biochemicals, Mannheim, Germany). Whole-cell lysates were incubated on ice for 30 minutes and clarified by means of centrifugation. Whole-cell lysates were eluted with SDS-PAGE sample buffer, resolved on Novex 4-12% SDS-PAGE gels with MES running buffer, and subsequently transferred onto nitrocellulose membranes. Membranes were blocked overnight in 3% BSA plus 0.1% Tris-buffered saline–Tween 20 at room temperature for 1 hour and then probed overnight at 4°C with primary antibodies, including α-NLRC4 (rabbit α-NLRC4; D5Y8E; Cell Signaling Technology, Danvers, Mass), α–caspase-1 (mouse α–caspase-1 p20; AdipoGen, San Diego, Calif), α-ASC (rabbit α-ASC; sc-22514; Santa Cruz Biotechnology, Dallas, Tex), α–caspase-8 (mouse α–caspase-8; #9746; Cell Signaling Technology), and α-actin (goat α-actin; sc-1616; Santa Cruz Biotechnology).

Sup, Lys, and Pellet samples were separated by SDS-PAGE (10% or 16%) using running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3) and Mini-PROTEAN® Tetra Handcast Systems (BIO-RAD, Hercules, CA, USA) and transferred onto a polyvinylidene difluoride membrane (PVDF; 10849A, Pall Co., Port Washington, NY, USA) using transfer buffer (25 mM Tris, 192 mM glycine, 10% methanol, pH 8.3) and Criterion^TM Blotter (BIO-RAD). The membrane was probed with primary antibodies against anti-mouse IL-1β antibody (AF-401-NA, R&D Systems, Minneapolis, MN, USA), anti-Caspase-1(p20) antibody (AG-20B-0042-C100, AdipoGen^® Co., San Diego, CA, USA), anti-Asc antibody (sc-22514, Santa Cruz Biotechnology), anti-NLRP3 antibody (AG-20B-0014-C100, AdipoGen^® Co.), anti-GSDMD antibody (ab209845, Abcam plc., Cambridge, MA, USA), or anti-Actin antibody (sc-1615, Santa Cruz Biotechnology) overnight at 4 °C. The membranes were further probed with HRP-conjugated 2^nd anti-sera (sc-2020 or sc-2004, Santa Cruz Biotechnology) and visualized using a chemiluminescence detection solution (WESTSAVER STAR, AbFrontier, Seoul, Republic of Korea) and a cooled CCD camera System (AE-9150, EZ-Capture II, ATTO Technology, Tokyo, Japan). Full-length bolts for figures were shown in Supplementary information.