2.5. Western Blotting for NLRP3-Inflammasome Expression Levels

Total proteins were extracted from lysates of THP-1 cells treated with plasma from active AOSD patients or healthy controls. The samples were run on 10% SDS-PAGE and then transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). The blots were blocked with 5% milk in PBS with 0.1% Tween-20 (PBST) (Bionovas, Inc., Washington, DC, USA) for 30 min at room temperature, and subsequently incubated with specific anti-CLEC5A antibody (Aviva Systems Biology, San Diego, CA, USA), anti-NLRP3 antibody (Cell Signaling Technology, Beverly, MA, USA), anti-caspase-1 antibody (Abcam, Cambridge, MA, USA), anti-IL-1β antibody (Novus Biologicals, LLC, Littleton, CO, USA), anti-IL-18 antibody (Medical & Biology Laboratories Co, Ltd., Naka-ku, Nagoya, Japan), and anti-α-tubulin (1: 5000, Santa Cruz Biotechnology, Dallas, Texas, USA) at 4°C overnight. After washing with PBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (Thermo Fisher Scientific Inc., Pittsburgh, PA, USA). Immunoreactive bands were incubated with an ECL detection system (Advansta, Menlo Park, CA, USA) and visualized by radiographic film. The band intensity was quantitated by ImageJ software as described previously [22]. The protein levels of NLRP3, caspase-1, IL-1β, and IL-18 were normalized to α-tubulin.