Western Blot Analysis of Inflammasome Proteins

Kidney and cell samples were lysed using the RIPA lysis buffer, and the nuclear proteins were extracted using a nuclear protein extraction kit. The protein concentrations of the lysates were determined using a BCA Protein Assay Kit. Next, 20 µg total protein per sample was resolved using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto 0.2 µm PVDF membranes by using a semidry Trans-Blot apparatus (BIO-RAD, Hercules, CA, USA). Subsequently, the membranes were blocked at room temperature (20–30 °C) for 1 h with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 (TBST). Afterward, the membranes were incubated overnight at 4 °C with the following primary antibodies: antibodies against NLRP3, cleaved caspase-1, cleaved IL-1, caspase-1, ASC, IL-33, ST2, NF-κB, tubulin β, lamin B, and GAPDH. Next, the membranes were washed three times with TBST and then incubated at room temperature for 2 h with a secondary anti-rabbit or anti-mouse antibody, as required. The blots were analyzed using an Odyssey fluorescence scanner (LI-COR, Biosciences, Lincoln, NE). The signals were captured by using the supplied Odyssey software v3.0, and the results were expressed as fold changes normalized to the expression levels of tubulin, GAPDH, or lamin B.