E cadherin

After lysing the cells, the protein concentration was assessed utilizing the BCA Quantitation Kit (Thermo). Subsequently, an equal amount of protein was resolved by 6% SDS-PAGE and transferred onto a nitrocellulose filter membrane. At room temperature, the membrane was treated in 3% skim milk for 2 h to block non-specific binding. In this study, due to multiple antibodies incubated at the same time will interfere with each other, resulting in mixed bands, the membrane is cut and underwent overnight incubation at 4 °C with specific primary antibodies respectively: BAZ2A (ab290639, Abcam, Cambridge, MA, USA), GADPH (ab8245, Abcam), BAX (ab32503,Abcam), Snail (MA5-14801, Thermo), p53 (GTX34938, GeneTex, TX, USA), Bcl-2 (GTX100064, GeneTex), Vimentin (GTX40346,GeneTex), E-cadherin (CSB-RA576116A0HU, CUSABIO, Wuhan, China), and N-cadherin (CSB-RA243509A0HU, CUSABIO). The membrane was subjected to 1 h of incubation with the corresponding secondary antibody (CSB-PA564648/CSB-PA573747, CUSABIO). ECL chemiluminescence substrate (PerkinElmer, Waltham, MA, USA) was used to detect bands.

Flow cytometry was utilized for the purpose of determining the percentage of Th9 cells present in intestinal tissue or serum [Donkey anti-sheep antibody Alexa fluor647 (Abcam, ab150179), FITC-CD4 antibody (eBioscience, 11-0400-82), IL-9 antibody (eBioscience, PA5-47584)]. The enzyme-linked immunosorbent assay (ELISA) kits were utilized for the detection of the serum levels of αEβ7 (MyBioSource, San Diego, CA, USA), E-cadherin (Cusabio, Wuhan, China), IL-9 (Cusabio, Wuhan, China), as well as D-lactate (a barrier function biomarker) (MyBioSource, San Diego, CA, USA).