Anti cathepsin b

LPS (L3012, L4391), ATP, nigericin, poly(I:C), poly(dA:dT), PI, SR9009, and SR8278 were purchased from Sigma‒Aldrich. Recombinant mouse serum amyloid A1 and recombinant mouse IFN-β were obtained from R&D Systems. Flagellin was purchased from InvivoGen. LLME was purchased from Cayman. SAA1 ELISA kits were purchased from R&D Systems. The following antibodies were used in this study: anti-caspase-1 (AG-20B-0042, Adipogen), anti-NLRP3 (AG-20B-00140, Adipogen), anti-mouse IL-1β (AF-401-NA, R&D Systems), anti-caspase-11 (NB120-10454, Novus Biologicals), anti-Bmal1 (93806, Abcam), anti-GSDMD (209845, Abcam), anti-cathepsin B (AF965, R&D Systems), anti-cleaved caspase-3 (9579, Cell Signaling Technology), anti-fibrin (MVI-1007, MERU-VasImmune), anti-phospho-STAT1 (#7649, Cell Signaling Technology), anti-STAT1 (#14994, Cell Signaling Technology), anti-β-actin (sc-47778, Santa Cruz Biotechnology), anti-LAMP-1 (sc-19992, Santa Cruz Biotechnology) and anti-C/EBPβ (sc-7962, Santa Cruz Biotechnology).

A253 cells were lysed in RIPA Lysis and Extraction Buffer with protease and phosphatase inhibitors (all from Thermo Fisher Scientific) and cleared by centrifugation at 17,000 g at 4°C for 25 minutes. Supernatants were heated at 97°C in NuPAGE LDS Sample Buffer for 10 minutes, resolved by SDS-PAGE, and electrophoretically transferred to polyvinylidene difluoride membranes (all from Thermo Fisher Scientific). Membranes were blocked with 2% non-fat dried milk at 25°C for 1 hour, and then incubated at 4°C overnight with one of the following primary antibodies: anti-cathepsin B (#AF953, R&D system, USA), anti-LAMP1 (#21997-1-AP, Proteintech, USA) or anti-α-tubulin (#T6199, Sigma-Aldrich, USA). After washing three times, membranes were incubated with rabbit or mouse IgG horseradish peroxidase-linked whole antibody (Sigma-Aldrich) at 25°C for 1 hour. Signals were visualized using Super Signal West Pico Chemiluminescent Substrate or Super Signal West Pico PLUS Chemiluminescent Substrate (both from Thermo Fisher Scientific).