Anti myd88

The proteins were extracted with RIPA lysis buffer and PMSF; then, the protein quantification was conducted with a BCA protein quantification kit. Processed protein samples were collected for 8–10% SDS-PAGE electrophoresis and transferred on PVDF membranes. The membranes were blocked with 1× TBST containing 5% skimmed milk powder for 1 h at room temperature. After membrane washing, the membranes were incubated with the primary antibodies listed below at a temperature of 4 °C overnight: anti-TLR4 (1:1000, Protein-tech, Wuhan, China), anti-NF-κB (1:1000, Protein-tech, Wuhan, China), anti-MyD88 (1:1000, Protein-tech, Wuhan, China), anti-AQP1 (1:1000, Protein-tech, Wuhan, China), anti-HO-1 (1:1000, CST, Boston, MA, USA), anti-Nrf2 (1:1000, Protein-tech, Wuhan, China), and anti-β-actin (1:1000, Affinity, Changzhou, China). The membranes were then incubated with horseradish peroxidase (HRP)-labeled goat anti-rabbit/mouse antibodies (1:3000, protein-tech, Wuhan, China) for 1 h at 37 °C, and the membranes were rinsed 6 times with 1× TBST to remove any remaining antibodies (5 min every time). The images were captured using an Automatic Electrophoresis Gel Imaging System (Tanon 5200 Multi, Shanghai, China).

The Western blot protocol used was previously described [7 (link)]. The primary antibodies included anti-TLR4 (Proteintech, China), anti-MyD88 (Proteintech), anti-NF-kB (R&D Systems, U.S.A.), anti-RIPK3 (Proteintech), anti-MLKL (Proteintech), anti-NLRP3 (ZEN BIO, China), anti-Caspase1 (Novus, U.S.A.), anti-Caspase8 (Proteintech), anti-GSDMD (ABclonal, China), anti-IL-1β (R&D Systems), and anti-β-actin (Elabscience, China). An anti–rabbit (or goat or mouse or rat) antibody (ABclonal) were applied to the secondary antibody.

Radio‐immune precipitation assay lysis buffer (Beyotime, P0013B) was used for tissue and cell lysates. BCA assay kit (Beyotime, P0012S) was used for protein concentration determination. After 30 µg protein amount was separated using 4%–20% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, it was transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% skim milk at 37°C for 2 h and then incubated with primary antibodies at 4°C overnight and secondary antibodies at 37°C for 1 h. After washing, the membranes were visualized by an ECL Kit (MedChemExpres) and quantified with ImageJ software. All experiments were repeated in triplicate for statistics. Following primary antibodies were used: anti‐iNOS (1:1000, Proteintech), anti‐COX‐2 (1:1000, Proteintech), anti‐IL‐6 (1:1000, Proteintech), anti‐TLR4 (1:1000, Proteintech), anti‐MYD88 (1:1000, Proteintech), anti‐NF‐κB‐p65 (1:1000, Affinity), anti‐NF‐κB‐p‐p65 (1:1000, Affinity), anti‐CD74 (1:1000, Abcam), β‐Actin (1:2000, Abcam).