Mini protean tetra cell and mini trans blot electrophoretic transfer cell system

Total protein extraction was performed using the M-PER mammalian protein extraction reagent (Thermo Fisher Scientific, Inc.). The concentration of proteins was measured with the bicinchoninic acid (BCA) assay kit (Sigma-Aldrich). Western blot analysis, including gel electrophoresis and transfer, was conducted by means of the Mini-PROTEAN Tetra Cell and Mini Trans-Blot electrophoretic transfer cell system (Bio-Rad Laboratories, Hercules, CA, USA)^{15 (link)}. The membranes were immunoblotted with specific primary antibodies (1:500–1000) against p-p65, p65, p-JNK, JNK, p-IRF3, IRF3, p-TBK1, TBK1, p-ERK, ERK, p-p38, p38, Iκ-Bα, and human IL-1β (Cell Signaling Technology Inc., Danvers, MA, USA); ATF3, COX2, caspase 1, and β-actin (Santa Cruz Biotechnology Inc., Dallas, TX, USA); iNOS (BD Biosciences); IL-6 and mouse IL-1β (R&D Systems Inc.); TNF-α (Thermo Fisher Scientific, Inc.); or NLRP3 (Adipogen, San Diego, CA, USA) with gentle shaking at 4 °C overnight. Next, the membranes were rigorously washed with PBST and incubated with a peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin G antibody (1:1000) for 2 h. The proteins were detected by means of a SuperSignal West Pico ECL solution (Thermo Fisher Scientific, Inc.) and visualized on a Fuji LAS-3000 system (Fujifilm, Tokyo, Japan) or ChemiDoc Touch Imaging System (Bio-Rad Laboratories).