Hrp conjugated secondary antibodies against rabbit or mouse igg

BMDCs, peritoneal macrophages, or peritoneal neutrophils were lysed with 1X CST lysis buffer (Cell Signaling). For neutrophils, DFP (Sigma Aldrich) was added to the lysis buffer to inhibit any protease activity. BCA assay kit (Thermo Fisher) was used to determine protein concentration from lysates. Twenty μg of protein from lysates mixed with 1X SDS (from 5X stock), and Ultrapure water (Invitrogen) were boiled for 10 minutes at 95°C on a heating block. Samples were loaded into 4%–20% mini-PROTEAN, 10-well, 50 μl TGX precast SDS-PAGE gels (Bio-rad). Gels were run in 1X TAE buffer at a constant 110V. Proteins were transferred onto a nitrocellulose membrane using Bio-rad Trans-blot Turbo transfer system. The membrane was blocked with 5% milk for 1 hour at RT, and incubated with rabbit anti-mouse GSDMD (EPR20859, Abcam) or mouse anti-β-actin (Santa Cruz Biotechnology) diluted in 5% milk and incubated at 4°C overnight on a rocker. Membranes were washed with 1X TBST buffer 3× for 10 minutes. HRP-conjugated secondary antibodies against rabbit or mouse IgG (Cell Signaling) were diluted in 5% milk and incubated at RT for 1 hour. West Femto Maximum Supersignal (Thermo Fisher) was used to enhance signal before the membrane was imaged by the Chemidoc (BioRad) instrument.