Cytoplasmic Fractionation and Western Blotting

Stimulated cells and tissue samples were harvested for protein extraction and western blotting analysis with standard protocols as previously described^{12 (link),56 (link)}. Cytoplasmic fractionations were performed with NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, Rockford, IL, USA) according to the manufacturer’ s protocol. Briefly, cells were harvested, washed and pelleted. Then added ice-cold CER I to the cell pellet and incubated the tube for 10 min on the ice. After that, added ice-cold CER II to the tube and incubated for 1 min on the ice. The volume ratios of cell pellets, CER I and CER II are 10: 100: 5.5. At last, centrifuged the tube for 5 min at 16, 000 × g and transferred the supernatant to a clean pre-chilled tube. Stored this tube in −80 °C until use. The following antibodies were used: anti-GAPDH (1:10000; Genetex, Irvine, CA, USA); anti-DAPK1, anti-MLC (1:1000; Abcam, Cambridge, MA, USA); anti-p-MLC (1:500; Cell Signaling Technology, MA, USA); anti-IL-1β (1: 2000; R&D Systems, Wiesbaden, Germany); anti-NLRP3, anti-caspase-1, anti-ASC (1:1000; Adipogen, San Diego, CA, USA)]; anti-cathepsin B (1:300; Santa Cruz Biotechnology, Santa Cruz, CA, USA); secondary antibodies (1:400; Jackson ImmunoResearch Laboratories, PA, USA). The intensity of protein bands was analyzed with the Image J software (NIH) and normalized to GAPDH.