Western Blot Analysis of Inflammasome Proteins

Cell proteins were extracted using RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.) with a protease inhibitor cocktail (Beyotime Institute of Biotechnology). First, equal amounts of proteins (50 µg) were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis and then transferred onto polyvinylidene fluoride membranes. Next, 5% fat-free milk was used to block the membranes for 2 h, which were then incubated with primary antibodies: IL-1β (#12242, Cell Signalling Technology, CST), cleaved IL-1β (#52718, CST), IL-18 (ab71495, Abcam) 1:1000; caspase-1 (#24232, CST), cleaved caspase-1 (#89332, CST), NLRP3 (ab214185, Abcam), NEK7 (ab133514, Abcam) 1:500; ASC (#67824, CST) 1:200; and GAPDH (#5174, CST) 1:2000, overnight at 4 °C. Then the blots were incubated with secondary antibodies for 2 h. Finally, protein bands were visualized with an enhanced chemiluminescence (ECL) system (KeyGEN, Nanjing, China) and scanned with a chemiluminescence imaging system (Gel Catcher 2850, China). The grey values of bands were analysed with NIH ImageJ v1.52 software^{61 (link)}.