W063 1 1

The total protein was extracted from mouse kidney tissues or cells using radioimmunoprecipitation assay lysate (W063-1-1, Jiancheng) [34 (link)]. After the protein concentration was measured using the bicinchoninic acid kits (P0012S, Beyotime), the proteins (40 μg) were isolated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride membranes. Next, the membranes were blocked with 5% skim milk for 1 h and incubated with rabbit primary antibodies anti-Nephrin (1:1000, ab216341, Abcam), anti-Podocin (1:10000, ab181143), anti-Nrf2 (1:1000, ab92946), anti-heme oxygenase-1 (HO-1) (1:1000, ab68477), anti-NLRP3 (1:1000, ab270449), anti-ASC (1:5000, ab155970), anti-Pro Caspase-1 (1:200, ab238972), anti-Gasdermin D N-terminal domain (GSDMD-N) (1:1000, ab215203), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1000, ab9485) overnight at 4 °C. After washing with PBS, the membranes were incubated with HRP-labeled goat anti-rabbit secondary antibody IgG H&L (1:20000, ab97051) at room temperature for 30 min. After that, the membranes were developed by enhanced chemiluminescence and then observed and photographed. Image-Pro Plus 6.0 (Media Cybernetics, Inc., MD, USA) was adopted to analyze the relative expression of different proteins, with GAPDH as an internal reference.

Following previous work [34 (link)], kidney tissue homogenate or MPC5 cells were collected. Total protein was extracted using radioimmunoprecipitation assay lysate (W063-1-1, Jiancheng Bioengineering Institute, Nanjing, China). Protein concentration in the supernatant was measured using the bicinchoninic acid method (P0012S, Beyotime). Subsequently, the supernatant was diluted 20 times and 50 μL of diluted supernatant was collected to detect the content of the target protein. The levels of OS-related indexes malondialdehyde (MDA) (A003-1-2), superoxide dismutase (SOD) (A001-3-2), and glutathione (GSH) (A006-1-1) were detected by kits (Jiancheng). The mouse serum or MPC5 cells were collected, followed by determining the levels of inflammatory cytokines interleukin (IL)-1β (H002) and IL-18 (H015) by ELISA kits (Jiancheng).