Keap1 antibody

The HTF was obtained by the method of Cheng et al. with some modifications. 95% ethanol (analytical-grade) was purchased from the Chengdu Chron Chemicals Co., Ltd. (Chengdu, Sichuan, China). The AB-8 macroporous resin was purchased from Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Cytokines in serum and liver (interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α)) were measured using commercial kits purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Both serum and liver biochemical indicators (high-density lipoprotein (HDL-C), low-density lipoprotein (LDL-C), triglyceride (TG), total cholesterol (TC), glutamic oxalacetic transaminase (GOT), glutamic pyruvic transaminase (GPT)) were measured using Nanjing Jiancheng commercial kits. Antibodies in the experiment were purchased from Proteintech Group, Inc. (Wuhan, Hubei, China), including the Nrf2 antibody, HO-1 antibody, TLR4 antibody, MyD88 antibody, GAPDH antibody, and Keap1 antibody.

Cells were washed with ice‐cold phosphate‐buffered saline (PBS) and lysed in RIPA buffer (150 mM NaCl, 1% NP‐40, 50 mM Tris/HCl (pH 8.0) and 10% glycerol) containing protease and phosphatase inhibitors purchased from Selleck. Cell debris was removed by centrifugation at 12 000 rpm for 20 minutes at 4°C. The protein concentration of the whole cell lysate was measured using a Thermo Pierce BCA Protein Assay kit. Equal amounts of total protein were separated with SDS‐PAGE and then transferred to PVDF membranes. Antibodies against dCK and NRF2 were purchased from Abcam. The Keap1 antibody was obtained from Proteintech.

Losartan potassium tablets were purchased (Merck Sharp & Dohme (Australia) Pty., Ltd, South Granville, Australia, import drug registration nos. H20160398 and H20160401); streptozocin (STZ) was purchased from Sigma Chemical Co., St. Louis, USA, CAS no. 18883-66-4; Keap1 antibody was bought from Proteintech Group, Wuhan, China, no. 13161-1-ap; Nrf2 antibody was purchased from Proteintech Group, Wuhan, China, no. 16396-1-ap; heme oxidase-1 (HO-1) antibody was purchased from Proteintech Group, Wuhan, China, no. 10701-1-ap; the malondialdehyde (MDA) test box was purchased (Nanjing Jiancheng Bioengineering Institute, Nanjing, China, no. A003-1). Acetonitrile was purchased from Merck, Billerica, USA; asiaticoside reference was purchased from the National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China; astragaloside reference was purchased from the National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China; triptolide reference was purchased (content ≥98%, the National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China).

Nitrosylation was detected using the S-nitrosylation assay kit (Thermo, 90,105). The protein was enriched using a Keap1 antibody (Proteintech, Cat#10503-2-AP) cross-linked with magnetic beads. Next, the protein was extracted, and the protein sample concentration was adjusted to 1 mg/ml using lysate. Next, half of the total was combined with five times the loading buffer and heated at 95 °C for 10 min. The other half was added to a corresponding volume of MMTS and vigorously shaken to block free sulfhydryl groups, precipitate the protein and remove excess MMTS. It was then resuspended using HENS buffer, and sodium ascorbate was added to reduce S-nitrosocysteine. Then, it was labeled with iodoTMT reagent, and Western blot detection was performed using an anti-TMT antibody. The control group will have the addition of corresponding DMSO, labeled as VEH.