Tgx precast sds page gels

Total protein was isolated from cells using RIPA lysis buffer (Thermo Fisher Scientific) and Halt protease inhibitor cocktail (Thermo Fisher Scientific). Protein concentration was measured using the Pierce BCA protein assay kit (Thermo Fisher Scientific) and cell lysates containing 20 µg of total protein were loaded and separated in 4-20% TGX precast SDS-PAGE gels (Bio-Rad, Hercules, CA) followed by transfer onto PVDF membranes (Bio-Rad). Immunoblot analyses were carried out as previously described (19 (link)). Mouse monoclonal antibody against GFP (1:1000, Santa Cruz, Dallas, Texas) and HRP-conjugated mouse monoclonal antibody against β-actin (1:50,000, Sigma-Aldrich, St. Louis, MO) were used for the assay.

Western blot analysis was performed essentially as described by Verbrugge et al.^{[22 (link)]}. Briefly, cell lysates were prepared from 5 × 10⁶ cells suspended in 150 µL ice-cold lysis buffer (Cell Signalling Technology, #9803) containing 4% Protease Inhibitor Cocktail (PIC) and 1 mM NaVO₄. Supernatant fractions were collected by centrifugation (13,000 × g for 10 min, 4 ^oC), and 30 μg protein aliquots were resolved on a 4%-20% TGX pre-cast SDS PAGE gels (Bio-Rad), followed by transfer onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA) suitable for chemiluminescent detection by the Odyssey Infrared Imaging System (PerkinElmer, Zaventem, Belgium). The membranes were pre-incubated in blocking buffer (Odyssey Blocking Buffer, LI-COR, Biosciences, Nebraska, USA) for 1 hr. Next, membranes were incubated overnight (4 ^oC) with primary antibodies and β-actin for control of equal loading. After three washing steps (PBS/0.05% Tween20), the membranes were incubated (1 hr) with secondary antibodies, followed by antibody detection with the LI-COR Odyssey scanner (Biosciences) and digital image acquisition/quantification with the Odyssey infrared imaging system software (version 3.0.16, LI-COR Biosciences) according to the manufacturer’s instructions.

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.