Anti nlrp3 antibody

All the materials were obtained from the suppliers as follows: PBS (Lot#8122153) (Gibco, NY, United States); uric acid (Lot#BCCC5658) (Sigma-Aldrich, Darmstadt, GER); Etoricoxib tablets (Lot#U039558) (J20180059) (Merck and Co., Inc., NJ, United States); WSP (Lot#2205001) (Z51020967) (Jiuzhaigou Natural Pharmaceutical Group Co., Ltd., Aba, CHN); 2-Chlorophenylalanine (Lot#20211126), Ammonium formate (Lot#T2122203) (Aladdin, Shanghai, CHN); Acetonitrile (Lot#R142221) (Dikma, CA, United States); formic acid (Lot#20221214) (TCI, Shanghai, China); Anti-IL-1β antibody (Lot#BB09202951), Anti-p-NF-κB P65 antibody (Lot# BB05301615), and Anti-NLRP3 antibody (Lot#BA12271673) (Bioss, Beijing, CHN).
Preparation of MSU suspension: A total measurement of 168.1 mg of uric acid and 818.6 mg of NaCl was dissolved in 100 mL of boiling water, and 1.25 mL of 1 mol/L NaOH was added, heated, and stirred until fully dissolved. The solution was left at room temperature for 2–3 days. The MSU crystal was obtained after filtering and drying and was sterilized at 180°C for 2 h. Subsequently, it was weighed in a clean area and prepared with sterile PBS as an MSU suspension (12 mg/mL).

BRL cells were exposed to H₂O₂ with or without L-NAT, fixed in 4% paraformaldehyde for 15 min and permeabilized with 0.3% Triton X-100-PBS for 15 min. Blocking was done in 5% goat serum for 30 min at 37 °C to inactivate endogenous peroxidase. Subsequent, the cells were incubated with anti-ASC antibody (1:200; Bioss), anti-NLRP3 antibody (1:200; Bioss), anti-IL-1β antibody (1:200; Bioss), anti-Caspase-1 antibody (1:100; Santa), anti-TLR4 (1:500; Bioss), and anti-NF-κB (1:500; Bioss) at 4 °C overnight and then incubated with FITC-conjugated secondary antibodies (1:150) at 37 °C for 30 min. DAPI staining was performed and image were taken using confocal microscopy (Olympus FV500; Olympus, Tokyo, Japan). Liver tissue from the sham group, I/R group and I/R + L-NAT group were made frozen sections of 10 µm thickness. And then immunofluorescence staining was performed using the method described above.

For protein extraction, each of 100 mg liver tissues were homogenized on ice in 1 ml RIPA lysis buffer with protease inhibitor mix PMSF. Lysates were obtained by centrifugation at 15,000 rpm for 15 min at 4 °C. The protein concentration was quantified by BCA method. Subsequently, protein samples were subjected to 10% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane, and then incubated with the following antibodies: anti-ASC antibody (1:200; Bioss), anti-NLRP3 antibody (1:200; Bioss), anti-TLR4 (1:500; Bioss) and anti-NF-κB (1:500; Bioss). Finally, the bands were observed using chemiluminescence (ECL) system according to the manufacturer’s instructions. Western blotting bands were analyzed using optical density scanning and Image Lab software (Bio-Rad). GAPDH (1:1000; Proteintech) was used as the loading control.