Il 38

For confirmation of the native folding and quantitation analyses, protein-specific ELISA experiments with the plantakines IL-37b (Invitrogen™, Cat. No. LS885210322) and IL-38 (R&D Systems Inc., Cat. No. DY9110-05) were performed according to manufacturers’ recommendations. BioTek Instruments (VT, USA) Epoch Microplate Spectrophotometer was used to acquire numerical ELISA data.

Serum of mice was obtained from peripheral blood extracted 24 h after CLP. Levels of activity or concentration of the following analytes were determined using ELISA kits following the manufacturer's instructions: aspartate aminotransferase (AST), creatinine, and alanine aminotransferase (ALT) (Sigma-Aldrich), as well as IL-38 (R&D Systems), IL-10 (ExCell Biology, Shanghai, China), TNF-α (ExCell Biology), IL-6 (ExCell Biology), and IL-1β (ExCell Biology). Plates were analyzed using a microplate reader (Tecan, SPARK 10 M, Austria).

The protein expression levels of target genes were detected by western blot. Total proteins were extracted from heart tissues or cultured cells using RIPA lysis buffer (Beyotime) according to instructions provided with the kit and protein concentrations were measured using a BCA Protein Assay Kit (Pierce Biotechnology Inc.). Antibodies used in this study are as follows: glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) (Immunoway), p65NF‐κB (R&D Systems), NLRP3 (Cell Signaling Technology), and IL‐38 (R&D Systems). Equal amounts of denatured protein samples were separated on 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis prepared in advance for about 2 h and then were transferred to polyvinylidene difluoride membranes, followed by blocking with 5% nonfat milk for 2 h and incubated with the indicated primary antibodies at 4°C overnight. After incubation with horseradish peroxidase‐conjugated secondary antibodies for about 2 h at room temperature, membranes were treated with ECL reagents (Thermo Scientific). GAPDH was used as the loading control to normalize comparisons, and data were analyzed using a densitometer.