Anti nlrp3 antibody

Myocardium tissues were obtained and lysed. Protein concentration was measured using a BCA protein assay kit. Then, protein samples were denatured by heating at 100 °C for 10 min. The protein samples were then separated by SDS-PAGE and transferred onto PVDF membranes. Thereafter, the membranes were incubated with corresponding primary antibodies overnight at 4 °C. The primary antibodies used in this experiment were anti-NLRP3 antibody (Cat. No.15101S, Cell Signaling Technology, 1:1,000), anti-caspase-1 antibody (Cat. No.22915-1-AP, Proteintech, 1:1,000), Caspase-1 Rabbit pAb (Cat. No. A0964, 1:1,000), HRP-conjugated β-actin rabbit mAb (Cat. No. AC028, 1:5,000) and anti-GADPH antibody (Cat. No.10494-1-AP, Proteintech,1:3,000). Then, the PVDF membranes were incubated with corresponding secondary antibody (Cat. No. SAA544Rb19, Cloud-Clone,1:5,000) for 2 hours at room temperature. The protein bands were visualized using ECL Western blotting substrate and photographed using C-DiGit 3600 (Li-Cor, United States of America).

Protein concentration was determined using a Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). Equal concentrations of proteins were mixed with SDS sample buffer and denatured at 95 °C for 5 min. The samples were resolved with 8% SDS–page gels which were then transferred onto nitrocellulose membranes. The membranes were blocked with 5% fat-free milk in 0.1% Tris-buffered saline with Tween (TTBS) for 2 hours and then probed with primary antibodies: anti-Nrf2 antibody (1:500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), anti-NLRP3 antibody (Cell Signaling Technology, Beverley, CA) at 4 °C overnight. After being washed for three times with TTBS, the membranes were incubated with secondary antibodies (Santa Cruz Biotechnology) in TTBS at room temperature for 2 hours. Membranes were washed again with TTBS three times and then visualized on X-ray films using a chemo-luminescence detection system (ECL, GE Healthcare). β-actin (Santa Cruz Biotechnology) and TATA box binding protein (TBP) (Abcam, Cambridge, UK) were used as protein loading control for cytoplasmic and nuclear proteins, respectively. The relative band intensities were measured by image analysis software Gel-Pro Analyser.

Cells were collected, trypsinized and lysed in RIPA lysis buffer. Transfer the electrophoresed proteins to a poly (vinylidene fluoride) membrane and incubate for 2 hours at room temperature in blocking solution. Then they were incubated in the membrane overnight at 4°C in antibody solution containing primary antibody, including anti-NLRP3 antibody and caspase-1 antibody (Cell Signaling Technology, USA). After overnight incubation, second antibody goat-anti-rabbit (1:5000) were added at 37 °C for 1.5h. The membrane was washed at room temperature for 30min and then detected with Amersham Imager 600 automatic chemiluminescence gel imaging analyzer.

Human macrophages were isolated as described before and seeded on 6‐well plates with 2.5 × 10⁶ cells per well. Stimulation was performed as described before. After stimulation, cells were washed two times with PBS, and afterward, 200 µl lysis buffer (1% Triton‐X, 150 mM NaCl, 50 mM Tris, 1% SDS, 0.5% deoxycholate) was added. Cells were scraped and incubated for 20 min on ice. Lysates were centrifuged at 20,000 × g for 20 min at 4°C. Afterward, lysates were diluted to 0.1% SDS with a dilution buffer (1% Triton‐X, 150 mM NaCl, 50 mM Tris, 0.5% deoxycholate). Next, lysates were incubated for 1 h at 4°C on a rotating wheel with anti‐NLRP3 antibody (1:200, Cell Signaling Technology). 50 µl protein‐agarose beads (Santa Cruz) were added per probe and incubated overnight at 4°C on a rotating wheel. Finally beads were washed, proteins were eluted from the beads and immunoblot was performed as described before.

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 (link)]. The protein levels of NLRP3, caspase-1, IL-1β, and IL-18 were normalized to α-tubulin.

Recombinant human SFRP-5 was obtained from R&D Systems (MN, USA). Isoproterenol hydrochloride was purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-Wnt5A antibody, anti-SFRP5 antibody, anti-IL-1 antibody, anti-IL-6 antibody, and anti-IL-18 antibody were purchased from Abcam (Cambridge, UK). An anti-glyceraldehyde-3 phosphate dehydrogenase (GAPDH) antibody was purchased from Bioworld (MN, USA). Anti-NLRP3 antibody and anti-JNK antibody were purchased from Cell Signaling Technology (Danfoss, MA, USA). Superoxide dismutase (SOD) and glutathione peroxidase (GSH-px) detection kits were purchased from Nanjing Jiancheng Institute of Biological Engineering (Nanjing, China).