Westsaver star

The Sup and Lys were separated by electrophoresis using 10 or 16% SDS-PAGE and transferred to a membrane (PVDF: GE HEALTHCARE BIO-SCIENCE, Pittsburgh, PA, USA)^{6 (link)}. The membranes were proved overnight at 4 °C as follows: anti- caspase-1 (p20) antibody (AG-20B-0042-C100, ADIPOGEN Co., San Diego, CA, USA), anti-GSDMD antibody (ab209845, ABCAM, Cambridge, MA, USA), anti-NLRP3 antibody (AG-20B-0014-C100, ADIPOGEN Co.), anti-mouse IL-1β antibody (AF-401-NA, R&D SYSTEMS), or anti-Actin antibody (sc-1615, SANTA CRUZ BIOTECHNOLOGY). The membrane was incubated with the secondary antibodies conjugated with horseradish peroxidase (INVITROGEN for anti-mouse antibodies, ABCAM for anti-gout antibodies) for 2 h at room temperature. Immunoblotting images were obtained using a chemiluminescent system (EZ-Capture II, ATTO TECHNOLOGY) and a chemiluminescence solution (WESTSAVER STAR, ABFRONTIER, Seoul, Republic of Korea). Supplementary information presents the full-length bolts for figures.

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.

Lys were separated by SDS-PAGE (10%) and transferred to a polyvinylidene difluoride membrane (PVDF; GE Healthcare Bio-Science, Pittsburgh, PA, USA). The membrane was incubated with the primary antibodies against anti-mouse LCN2 antibody (AF1857, R&D Systems, Minneapolis, MN, USA), anti-mouse IL-1β antibody (AF-401-NA, R&D Systems), or anti-Actin antibody (sc-1615, Santa Cruz Biotechnology, Dallas, TA, USA) overnight at 4 °C. The PVDF was further probed with 2nd anti-sera conjugated with horseradish peroxidase (donkey anti-goat IgG for LCN2 and IL-1β anti-sera, ab6885, Abcam, Cambridge, UK) and visualized using an enhanced chemiluminescence solution (WESTSAVER STAR, AbFrontier, Seoul, Korea) and a chemiluminescent system (EZ-Capture II, ATTO Technology, Tokyo, Japan).