Af7443

Total protein was extracted using RIPA lysis buffer, and the concentration of protein was determined using a BCA Protein Assay kit (Vazyme, Nanjing, China). After that, the protein was separated in a 12% SDS-PAGE gel and transferred to PVDF membranes (Immobilon; Millipore, Bedford, USA). The membranes were then incubated with 5% nonfat milk for 2 h at room temperature, followed by incubation with anti-RIP1 (1:1000, 17519-1-AP; Proteintech), anti-RIP3 (1:100, 15828; Cell Signaling Technology), anti-p-RIP3 (1:1000, AF7443; Affinity, Biosciences, Changzhou, China), and anti-p-MLKL (1:1000, AF7420; Affinity) antibodies overnight at 4°C. The next day, the membranes were incubated with HRP-conjugated goat anti-rabbit secondary antibody (1:5000, RGAR001; Proteintech) for 1 h at room temperature. Finally, an Enhanced Chemiluminescence kit (ChemiDoc XRS+; Bio-Rad, Hercules, USA) was utilized to visualize the protein bands, and the optical densities of the western blot bands were analyzed using Image-Pro Plus 6.0 software. The relative expressions of RIP1, p-RIP3, and p-MLKL in the ileum region were normalized to that of β-actin, while the relative expression of p-MLKL in the cell membrane of IEC-6 cells was normalized to that of Na/K ATPase, which was extracted using a Membrane and Cytosol Protein Extraction kit (P0033; Beyotime).