Tanon 6200 chemiluminescence imaging workstation

The cells were lysed in radioimmunoprecipitation assay (RIPA) buffer supplemented with a protease and phosphatase inhibitor cocktail (HY-K0010 and HY-K0022; MedChemExpress) on ice for 15 min. Protein samples were separated by SDS-PAGE and then transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). After being blocked in 5% skim milk (A600669; Sangon Biotech) at room temperature for 2 h, the membrane was incubated with the primary antibody overnight at 4°C and then with the appropriate HRP-conjugated secondary antibody for 1 h at room temperature. The target protein bands were detected with the ECL enhanced chemiluminescence detection system (Tanon 6200 chemiluminescence imaging workstation; Tanon Science & Technology Co., Ltd., Shanghai, China). The bands were then quantified by densitometry using ImageJ software.

Proteins were extracted from tissues (jejunum and ileum) or Caco2 cells using the RIPA buffer (Solarbio, Beijing, China) and 1 mM PMSF (Solarbio, Beijing, China). BCA Protein Assay kit (23227, ThermoFisher Scientific, Waltham, MA, USA) was used to quantify the protein concentration. Proteins were divided into 10% or 12% SDS-polyacrylamide gel electrophoresis and transferred to the polyvinylidene difluoride membranes (Roche, Basel, Swiss). Subsequently, proteins were blocked with 5% skim milk at 37 °C for 1 h and then were incubated with the following primary antibodies overnight at 4 °C: NOD1 (1:500), NOD2 (1:1000), RIP2 (1:1000), ATG16L1 (1:1000), IRGM (1:1000), P62 (1:1000), LC3A/B (1:1000), GRP78 (1:1000), IRE1 (1:1000) and EIF2S1 (1:1000). Secondary antibodies (1:5000) with corresponding species source were used to incubate at 37 °C for 1 h. The images were visualized by an ECL detection system (Tanon 6200 chemiluminescence imaging workstation, Tanon Science & Technology Co., Ltd. Shanghai, China).