Infrared imaging system

Harvested cells were lysed in IP lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 2 mM Na3VO4, 5 mM NaF, 1% Triton X-100) supplemented with protease inhibitor cocktail (Invitrogen) at 4 °C for 30 min. Then, the lysates were cleared by centrifugation at 12,000 × g for 10 min at 4 °C. The supernatant was immunoprecipitated with the indicated antibodies and protein G-agarose beads (Invitrogen). Then, standard WB analysis was performed. For WB analysis, cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (Beyotime Biotechnology, Jiangsu, China). Equal amounts of protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). The membranes were blocked in 5% milk for 2 h at room temperature and then incubated with the indicated primary antibodies overnight at 4 °C and HRP-conjugated secondary antibodies for 1 h at room temperature. The membranes were detected and analyzed using an infrared imaging system (Bio-Rad Laboratories, Inc.). The experiments were performed in triplicate.

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.