Bicinchoninic acid assay method

Tissues or cells were lysed with radioimmunoprecipitation assay lysate (catalog number: P0013B; Beyotime) to extract proteins whose concentrations were measured using the bicinchoninic acid assay method (catalog number: P0010; Beyotime). The protein samples (50 μg) underwent sodium dodecyl sulfate polyacrylamide gel electrophoresis; they were then transferred to polyvinylidene difluoride membranes (catalog number: IPVH00010; Millipore) and incubated with 50 mg/mL skimmed milk powder for 1 h. They were incubated with the primary antibody at room temperature for 1 h or overnight at 4°C. After incubating with the secondary antibody at room temperature for 1–2 h, enhanced chemiluminescence substrate solution (catalog number: P1050; APPLYGEN) was added dropwise to make the band appear. Grayscale analysis and statistics were performed using ImageJ software. Table 2 lists the antibody information.

Pancreatic tissue and pancreatic acinar cell extracts were used for western blotting analysis. Total amounts of protein were detected using the bicinchoninic acid assay method (Beyotime Biotechnology, China). Proteins (40 μg per lane) were separated by 10% SDS-PAGE at 120V and transferred to nitrocellulose membranes (Millipore, Mass, USA) for 30 ~ 60 min. Membranes were then incubated with primary antibodies against polyclonal GRP78 (1:1000), sXBP1 (1:400), RIP3 (1:400), MLKL (1:1000), IL-1β (1:1000), IL-6 (1:1000) and p-PKCα (1:400); monoclonal CHOP (1:1000), pMLKL (1:1000), CTSB (1:1000), p-JNK (1:1000), p-ERK (1:1000), p-p38MAPK (1:1000), p-cJun (1:1000), TNFα (1:1000) and β-actin (1:1000) overnight at 4°C. After being washed with PBS containing 0.1% Tween, membranes were probed by secondary antisera labelled with goat anti-mouse or goat anti-rabbit IR-Dye 700 or 800 cw for 1 h at 37°C. Membranes were scanned by an Odyssey Infra-red Scanner (LI-COR, Lincoln, NE, USA). Representative blot images were presented from three separate experiments. Relative expression of target proteins was expressed as fold changes compared to normal control after normalized to β-actin.