Il 18

Equivalent samples were separated via 10% SDS-PAGE, followed by transfer to PVDF membranes and blocking in 5% skimmed milk. Subsequently, the membranes were probed with primary antibodies against NLRP3 (Abcam), GSDMD-N (Abcam), caspase-1 (Cell Signaling Technology, Inc.), IL-1β (Abcam), IL-18 (Abcam), FTO (Cell Signaling Technology, Inc.), or GAPDH (Cell Signaling Technology, Inc.) at 4°C overnight, followed by treatment with the appropriate secondary antibodies. The protein bands were visualized using achemiluminescence system kit (Thermo Fisher Scientific, Inc.). The intensity of the bands was analyzed using ImageJ software. GAPDH served as an internal reference.

Protein levels of inflammasome signaling proteins were determined in the cytosolic and mitochondrial fractions as described in [7 (link)]. Briefly, protein lysates were resolved in 10–20% Criterion TGX Stain-Free precasted gels (Bio-Rad), using antibodies (1:1000 dilution) to NLRC4 (Novus Biologicals, cat# NBP2–41124), caspase-1 (Novus Biologicals, cat# NB100–56565), caspase-11 (Novus Biologicals, cat# MAB8648), ASC (Santa Cruz, cat# sc-271054), IL-1β (Cell Signaling, cat# 12242S), IL-18 (Abcam, cat# ab71495), HSP60 (Cell Signaling, cat#12165) and β-actin (Sigma Aldric, cat# A5441). Quantification of band densities was done using the UN-SCAN-IT gel 6.3 Software (Silk Scientific Corporation). Chemilluminescence substrate (LumiGlo, Cell Signaling) was imaged with the ChemiDoc Touch Imaging System (BioRad).

The lysates of kidney tissue and renal tubular epithelial cells were used to detect the proteins concentration. The standard procedure of Western blot was used as described, following with the used of these antibodies: Cx32 (1:1000; Abclonal); NLRP3(1:1000; Abcam); GSDMD (1:1000; CST); IL-18(1:1000; Abcam); β-actin (1:50000; Abclonal); Rabbit secondary antibody (1:20000; Abcam). All the Western blots were repeated for more than three times and the images were captured by Tanon 5500 imaging system (Tanon, Shanghai). All images were analyzed by the ImageJ scanning software and the level of proteins were expressed as the relative values to the sham or control groups.

The extraction of total myocardial tissue proteins was completed using RIPA lysis buffer (Solarbio, China), followed by the determination of protein concentration using a BCA kit (Solarbio, China). The proteins were denatured at high temperature, separated by SDS-PAGE electrophoresis, and then transferred to PVDF membranes (Millipore, USA). After completing the closure (60 min) at room temperature using non-fat milk, the proteins were incubated overnight at 4 ℃ with SIRT1 (dilution rate 1:1000, Abcam, USA), AMPK (dilution rate 1:1000, Abcam, USA), p-AMPK (dilution rate 1:1000, Abcam, USA), NLRP3 (dilution rate 1:1000, proteintech, China), GSDMD-N (dilution rate 1:1000, Cell Signaling Technology, USA), ASC (dilution rate 1:1000, Abcam, USA), IL-18 (dilution rate 1:1000, Abcam, USA), caspase-1 p20 (dilution rate 1:1000, Bioss, China), IL-1β (dilution rate 1:1000, Abcam, USA), and GAPDH (dilution rate 1:10000, Abcam, USA) primary antibodies. The proteins were incubated with secondary antibodies (dilution rate 1:10000, Santa Cruz Biotechnology, USA) at room temperature for 1 h the next day, and the protein bands were developed under the ultrasensitive automatic chemiluminescence imaging analysis system (FluorchemM, Protein Simple, USA). The detection of relative protein expression was completed using Image J (NIH, MD, USA) software.

Samples were homogenized in lysis buffer, and the supernatant was extracted for measurement of total protein with a Protein Assay Kit (BCA; Pierce, Santa Cruz, CA, USA). Equal amounts of total protein were separated in SDS-PAGE and transferred to PVDF membranes in Trisglycine methanol buffer. The bands were visualized using the enhanced chemiluminescence. GADPH was developed as a loading control to normalize the data. The antibodies against NLRP3, ASC, pro-caspase-1, IL-1β, Kim-1 and IL-18 were purchased from Abcam (Cambridge, MA, USA). Antibody against BCL6 was obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies against NGAL and Cystatin C were purchased from Bioworld Technology, Ltd (Nanjing, Jiangsu, China). Antibody against GAPDH was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Salidroside was provided by the Second Medical University (Shanghai, China; purity > 99%). MPTP-HCl was purchased from MedChemExpress (New Jersey, USA). Lipopolysaccharide (LPS) was purchased from Sigma Aldrich (St. Louis, USA). Enzyme-linked immunosorbent assay (ELISA) kits of interleukin (IL)-18 and IL-1β were purchased from Elabscience (Wuhan, China). The primary antibodies against MyD88, p-IκBα, IκBα, NF-κB, ASC, cleaved-Caspase-1, GSDMD, α-Synuclein, TH, TXNIP and GAPDH were purchased from Cell Signaling Technology (Danvers, MA, USA). The anti-p-NF-κB, TXNIP, NLRP3, IL-1β and IL-18 primary antibodies were produced by Abcam (Cambridge, UK). The anti-TLR4 primary antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The antibodies are listed in Supplementary Table 1 and the critical chemicals are listed in Supplementary Table 2.