Chemiluminescence kit

Cells were seeded in 6-well plates at a density of 5 × 10⁵ cells/well. After treatment with various concentrations (0, 10, 20, 40, and 80 µM) of IBC, the cells were harvested, washed twice with PBS, and homogenized in RIPA lysis buffer for 30 min on ice. Protein concentration was measured using the BCA protein assay kit according to the manufacturer’s protocol. Equal amounts of proteins were loaded onto 10~12% SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membrane was blocked using 5% skimmed milk and incubated with primary antibodies (Bcl-2, Bax, Akt, p-Akt-473, caspase-3, RIP1, RIP3, p-RIP3, MLKL, and LC3) overnight at 4°C, followed by the secondary antibodies at room temperature for 2 h. A chemiluminescence kit and gel imaging system were used to detect the protein bands (Bio-Rad, Hercultes, CA, USA). Anti- β-actin was used as an internal control.

Total proteins from CACO‐2 cells were treated with PC and RES, and their combinations were extracted using RIPA lysis buffer containing a protease and phosphatase inhibitor cocktail. Protein concentrations were quantified by BCA Protein Assay Kit (solarbio) and separated by SDS‐PAGE (10%) for electrophoresis and then transferred to a polyvinylidene difluoride (PVDF) membrane. Next, membranes were saturated with 5% non‐fat milk for 2 h at room temperature and then incubated with primary antibodies overnight at 4°C. Goat anti‐mouse and anti‐rabbit IgG conjugated with horseradish peroxidase (HRP) were used as secondary antibodies to incubate with the membranes for 1 h at room temperature. Immunoreactive bands were visualized using Chemiluminescence kit and determined by an infrared imaging system (Bio‐rad, United States). Quantitative analysis was conducted by Image J 1.53.

Protein samples from 3T3-L1 adipocytes were obtained using RIPA lysis buffer containing 1 mM phenylmethanesulfonyl fluoride (PMSF; FDbio, China). The total protein concentration was determined using the BCA method (FDbio, China). Equal amounts of proteins (40 μg per group) were loaded for SDS-PAGE electrophoresis. After transferring protein to polyvinylidene difluoride (PVDF) membranes, the blots were blocked with 5% skim milk for 1 h at room temperature. The membranes were incubated with primary antibodies overnight at 4°C and corresponding secondary antibodies conjugated with anti-rabbit IgG conjugated to horseradish peroxidase for 2 h at room temperature. All antibodies were obtained from Abcam (Waltham, MA, USA). β-Actin was used for normalization. Bands were visualized using a chemiluminescence kit (Bio-Rad, Hercules, CA, USA) and quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Cells for protein extraction were lysed with RIPA buffer and extracts were sonicated and centrifuged. Protein extracts were quantified with BCA kit (23,225, Thermo Fisher Scientific) according to the manufacturer’s protocol. 20 μg protein of samples was loaded in 12% polyacrylamide gels electrophoresis (PAGE). Electrophoresis was conducted at a constant amperage (30 mA) for approximately 2 h followed by the protein transference to a PVDF membrane at constant voltage (100 mV) for 1.5 h. 5% non-fat milk was used to block unspecific unions, and the primary antibodies mouse monoclonal anti-V-ATPase subunit V₀D₁ (ab56441, Abcam, used at 2.5 μg/mL) and monoclonal anti β-actin (a5316, SigmaAldrich, used at 1/20000), were incubated overnight. The next day, the secondary antibody was incubated for 1 h (goat anti-mouse IgG: 31430, Thermo Fisher Scientific, used at 1/10000). Finally, the membrane was developed using the chemiluminescence kit of BioRad according to the manufacturer’s protocol, and membrane chemiluminescence was detected with a Chemidoc MP Image System (BioRad). Image lab software (BioRad) was used for the quantification of bands.

Protein lysates were prepared from the thoracic aorta. Next, 5 µg of total protein for each sample was separated on a 10% Bis–Tris gel or 3–8% Tris–Acetate gel (Thermo Fisher Scientific) by SDS-PAGE, electroblotted onto nitrocellulose membranes using the iBlot2 Dry Blotting System (Invitrogen) and immunoblotted with primary antibodies. Membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies overnight and visualised with a chemiluminescence kit (Bio-Rad Laboratories).