Ecl developer

Cells were lysed using RIPA buffer (Beyotime Institute of Biotechnology), and the protein concentration was determined by using Pierce® BCA protein assay (Thermo Fisher Scientific, Inc.) using bovine serum albumin as a standard. Equal amounts of denatured proteins (30 μg) were subjected to SDS-PAGE electrophoresis and subsequently transferred to PVDF membrane (MiliporeSigma). After blocking the membranes with 5% defatted milk for 1 h, the membrane was incubated with the primary antibodies (see below) at 4°C overnight. After washing the membrane three times, the secondary antibody (see below) was added at room temperature for 1 h. Finally, the immunoblots were viewed using ECL Developer (Beyotime Institute of Biotechnology) and photographed with a gel imager (Bio-Rad Laboratories, Inc.). ImageJ software was used to calculate the relative gray values using β-actin as loading control. The antibody information was as follows: Primary antibody: anti-ALKBH5 antibody (1:1000, Cell Signaling Technology, Inc.), anti-MMP2 antibody (1:1000, Elabscience Inc.), anti-MMP9 antibody (1:1000, BOSTER Biological Technology Co. Ltd.), and anti-GAPDH antibody (1:5000, Beyotime Institute of Biotechnology); and secondary antibody: HRP conjugated anti-rabbit or -mouse secondary antibody (1:5000, MultiSciences Biotech Co., Ltd.).

The following Reagents and antibodies were used in this study: Dulbecco’s Modified Eagle Media: Nutrient Mixture F-12 (Hyclone, UT, USA); 1 × phosphate buffered saline (1 × PBS; Hyclone, UT, USA); Fetal bovine serum (ExCell Bio, Australian); Hydrogen peroxide (H₂O₂) (Chemistry Industry Co, Shandong, China); Cell Counting Kit-8 (Beyotime, Shanghai, China); ECL developer (Beyotime, Shanghai, China); caspase-1 inhibitor Ac-TYR-VAL-ALA-ASP-CMK (Ac-YVAD-CMK) (Cayman Chemical, MI, USA); lysosomal inhibitor bafilomycin-A1 (Aladdin, Shanghai, China); Primary antibodies: anti-NLRP3, anti-caspase-1, anti-IL-1β, anti-CTSD, and anti-GSDMD (Abcam, Cambridge, MA, USA); anti-keratan sulfate (5D4) (MD bioproducts, MN, USA); anti-GAPDH (Proteintect, IL, USA); anti-p62 and anti-LC3 (Cell Signaling Technology, MA, USA); TWEEN-20 (Solarbio, Beijing, China); HRP-conjugated secondary antibody (Zsbio, Beijing, China); LysoTracker Green DND-26 (Cell Signaling Technology, MA, USA).