0.2 μm nitrocellulose membranes

Twelve microliters of concentrated supernatant or lysate was separated on 4–12% Mini-Protean TGX gels (Bio-Rad, Hercules, CA, USA) at 100 V for 1 h. Proteins were transferred onto 0.2 μm nitrocellulose membranes (LiCor, Lincoln, NE, USA) at 100 V for 1 h, and subsequently blocked in 5% non-fat dry milk in PBS containing 0.2% Tween-20 for 1 h. Blocked membranes were incubated in 5% BSA in PBS containing 0.2% Tween-20 and either 1 : 500 rabbit polyclonal against Caspase-1 p10 (#SC-514, Santa Cruz) or 1 : 1000 goat polyclonal against IL-1β (#AF-401-NA, R&D Systems, Minneapolis, MN, USA) and rotated overnight at 4 °C. The following day, donkey anti-goat IRDye 800CW and goat anti-rabbit IRDye 680RD secondary antibodies (Li-Cor) were applied at a dilution of 1 : 15 000 with rocking for 1 h at room temperature. Membranes were imaged on a Li-Cor Odyssey CLx on auto exposure with high quality setting.

Twelve microliters of supernatant or cell lysate were separated on a 4–12% Mini-Protean TGX gel (Bio-Rad) at 100 V for 1 h. Proteins were transferred onto a 0.2 μm nitrocellulose membranes (Li-Cor Biotechnology) at 100 V for 1 h. The membranes were blocked in 5% non-fat dry milk in PBS containing 0.2% Tween-20 for 1 h followed by incubation in PBS containing 5% BSA, 0.2% Tween-20, and either 1:1,000 rabbit polyclonal anti-Caspase-1 p20 (47 (link)), anti-IL-1β (70 (link)), anti-HMGB1 (abcam; ab18256), anti-PKR (phospho T446) (abcam; ab32036), anti-PKR (abcam; ab226819), anti-beta Actin antibody (abcam; ab8229), Anti-GFP antibody (abcam; ab6556), or 1:500 anti-Asc (Tyr-144) phospho-specific antibody (ECM Bioscience; AP5631) with rotation overnight at 4°C. The following day, donkey anti-rabbit IRDye 800CW and donkey anti-goat IRDye 680RD secondary antibodies (Li-Cor Biotechnology) were applied at a dilution of 1:15,000 with rocking for 1 h at room temperature. Membranes were imaged on a Li-Cor Odyssey CLx machine with auto exposure and high-quality setting.