Epr19828

Laemmli buffer (5x) was added to samples and boiled at 95°C for 10 min before resolving by SDS-PAGE. Resolved gels were transferred onto nitrocellulose or PVDF membranes using a Trans-Blot^® Turbo Transfer^™ System (Bio-Rad). Membranes were blocked in 5% (w/v) milk in PBS 0.1% Tween-20 (PBST) for 1 h at room temperature. Membranes were washed with PBST and incubated overnight with rabbit-anti-mouse IL-18 (1/1000 dilution; E9P50, Cell Signalling Technology, 57058), rabbit-anti-mouse capase-1 p10 (1/1000 dilution; EPR16883, Abcam, ab179515), mouse-anti-mouse NLRP3 (1/1000 dilution; Cryo2, Adipogen, AG-20B-0014), rabbit-anti-mouse gasdermin-D (1/1000 dilution; EPR19828, Abcam, ab209845), or goat-anti-mouse IL-1β (1/800 dilution; R&D Systems, AF-401-NA), primary antibodies were diluted in 5% (w/v) BSA in PBST. The membranes were washed and incubated at room temperature for 1 h with rabbit anti-mouse IgG (Agilent, P026002–2) or goat anti-rabbit IgG (Agilent, P044801–2), secondary antibodies diluted 1/1000 in 5% (w/v) BSA in PBST. Proteins were then visualized with Amersham ECL Prime Western Blotting Detection Reagent (Cytiva, RPN2236) and G:BOX (Syngene) and Genesys software. β-Actin (1/20000 dilution 5% (w/v) BSA in PBST; Sigma, A3854) was used as a loading control.

Lysates and/or supernatants were assessed by western blot for NLRP3, IL-1β, caspase-1, and GSDMD. Samples were run on SDS polyacrylamide gels and transferred onto nitrocellulose or PVDF membranes using a semi-dry Trans-blot Turbo system (Bio-Rad) at 25 V. Membranes were blocked with 2.5% BSA in phosphate-buffered saline, 0.1% Tween 20 (Sigma) (PBST) for 1 h before overnight incubation at 4 °C with mouse anti-NLRP3 monoclonal antibody (Cryo2, Adipogen), goat anti-IL-1β polyclonal antibody (AF-401, R&D Systems), rabbit anti-caspase-1 + p10 + p12 monoclonal antibody (EPR16883, Abcam), or rabbit anti-GSDMD antibody (EPR19828, Abcam) in 2.5% BSA PBS-T. The following morning, membranes were washed (5 min, ×3) in PBST and subsequently incubated with either rabbit anti-mouse, rabbit anti-goat or goat anti-rabbit HRP antibodies (Dako) in 2.5% PBST for 1 h at room temperature. β-Actin was used as a sample loading control using a monoclonal anti-β-Actin-peroxidase antibody (Sigma). After washing, Amersham ECL prime detection reagent (GE Healthcare) was added to membranes and images were taken with a G:Box Chemi XX6 (Syngene) scanner.