Anti hmgb1

Total protein was extracted from the kidney tissues as previously described. Protein samples (50 μg/lane) were separated on a sodium dodecyl sulfate-polyacrylamide gel (12% gel) and transferred onto polyvinylidene fluoride membranes. The membranes were blocked in 5% nonfat milk for 1 h at room temperature and then incubated with primary antibodies on a rotating shaker overnight at 4 °C. The following primary antibodies were used: anti-β-actin (1:1000; cat. no. GB12001; Servicebio, Woburn, MA), anti-HMGB1 (1:2000; cat. no. GB11103; Servicebio, Woburn, MA), anti-Beclin 1 (1:1000; cat. no. GB13228-2; Servicebio, Woburn, MA), and anti-microtubule-associated protein 1 light chain 3, anti-LC3 (1:3000; cat. no. 14600-1-AP; Servicebio, Woburn, MA). Next, the membranes were washed three times with tris-buffered saline containing Tween20 (TBST), and then incubated with a horseradish peroxidase‑conjugated secondary antibody (1:3000; cat. no. GB23302, GB23303; Servicebio, Woburn, MA) at room temperature for 1 h. The membranes were subsequently washed three times with TBST and visualized using electrochemiluminescence. The Image Lab Analysis System (Alpha Innotech, alphaEaseFC, San Leandro, CA; Adobe, Adobe PhotoShop, San Jose, CA) was used to analyze the images.

Samples were lysed in RIPA buffer lysis containing protease inhibitors (PMSF) (Beyotime, China) at 4 °C, and used bicinchoninic acid (BCA) assay to determine the concentration. To further dilute the sample, 6× SDS-PAGE loading buffer was added and boiled (100 °C) for 10 min. An equal amount of the samples (20 μg) was acceded, electrophoresed on a 10% SDS-polyacrylamide denaturing gel, and then transferred to a polyvinylidene fluoride (PVDF) membrane. Anti-HMGB1 (1:1000; Servicebio, Wuhan, China), anti-p65 (1:1000; Bioss, Beijing, China), anti-p-p65 (1:1000; Sigma, Ronkonkoma, NY, USA), anti-TLR4 (1:1000; Sigma, USA), anti-MYD88, anti-NLRP3, anti-IL-1β, anti-pro-caspase-1 (1:500; Wanleibio, Shenyang, China), anti-ASC (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-GAPDH (1:5000; Bioss, China) as the primary antibody. With GAPDH as a reference, and the protein was measured and analyzed by Image J 1.48V and Image Lab 4.0 Software (Bio-Rad, Hercules, CA, USA).

Liver sections were stained with anti-CD68 (GB113109, Servicebio, Wuhan, China), anti-HMGB1(GB11103, Servicebio, Wuhan, China), or anti-myeloperoxidase (GB11224, Servicebio) according to a standard protocol. Apoptotic cells were evaluated using a TUNEL assay kit according to the manufacturer’s instructions (Servicebio, Wuhan, China). All the stained sections were observed under light microscopy, and images were collected by fluorescence microscopy with a Nikon Eclipse C1. For relative quantification of immunohistochemistry and immunofluorescent stained sections, three representative images from each section were analyzed.