Ab264468

Kidney and lung lysates were prepared for Western blot using the BioPlex Cell Lysis Kit (BioRad) according to the manufacturer’s protocol. A total of 100 μg of protein were separated by SDS-PAGE using a polyacrylamide gel (4–15%). The proteins were transferred onto 0.2 μm nitrocellulose membranes (BioRad). Membranes were blocked with blocking buffer (0.5% non-fat milk in 1x TBST) for 1 hour at room temperature and incubated overnight at 4°C with primary antibody directed against NLRP3 (1:1000; Recombinant anti-NLRP3 antibody, ab264468, Abcam) or Caspase-1 (1:1000; Recombinant anti-pro Caspase-1 + p10 + p12 antibody, ab238972, Abcam). After washing, the membranes were incubated with HRP-conjugated secondary antibody (1:2500; Goat anti-rabbit IgG H&L, ab205718, Abcam) for 1 hr at room temperature.The protein bands were developed with enhanced chemiluminescence reagents (BioRad) and visualized on a BioRad Chemidoc. The intensity of specific bands were quantified by densitometry using Image J (National Institutes of Health, Washington, DC) and expression was normalized with respect to β-actin expression (1:2000; Anti-β-actin antibody, a1978-100ul, Millipore Sigma, St. Louis, MO). Secondary antibody to β-actin: 1:2000; Goat anti-mouse IgG (H +L)-HRP conjugate, 1706516, BioRad.