Crhr2

There are two major types of immunofluorescent staining method: (1) Cells that attached to coverslips after experimental treatment were fixed with 4% paraformaldehyde for 20 min, and permeability with 0.2% Triton X-100 and blocked with PBS containing 5% BSA for 60 min in the next. The coverslips were followed by incubating with primary ZO-1/CRHR1/CRHR2, and labeled with specific secondary antibody for 1 h, and stained with DAPI (Biosharp, Hefei, China) for 5 min. The slides were washed with PBS and mounted by FluorSave^TM mounting media (Merck, Darmstadt, Germany). (2) The intestinal tissue was fixed in 10% phosphate-buffered formalin for immunofluorescent studies before paraffin embedding. For immunofluorescent staining of ZO-1 (1:500, Proteintech, Wuhan, Chian), CRHR1 (1:50, Proteintech, Wuhan, Chian), CRHR2 (1:200, Proteintech, Wuhan, Chian) and Tryptase (1:100, Santa Cruz, CA, USA), paraffin sections (5 μm) were dewaxed to rehydrate. After blocking with 10% goat serum, sections were incubated with primary antibody at 4 °C overnight, and detected with IgG (H + L) Highly Cross-Adsorbed Secondary Antibody (Thermofisher, Shanghai, China) for 1 h at room temperature, respectively. Representative images were captured under a fluorescence microscope and analyzed with imageJ Software (ImageJ 1.46r, National Institutes of Health, Bethesda, MD, USA).

After drug treatment, cell/small intestine tissue was harvested and lysed with 1 × RIPA lysis buffer (Beyotime, Shanghai, China) containing phenyl-methyl-sulfonyl fluoride (PMSF, Beyotime, Shanghai, China). The proteins were obtained from cell lysates and separated on SDS-PAGE. After transferring proteins to PVDF membranes (Milipore, Darmstadt, Germany), the membranes were blocked with TBST supplemented with 5% skimmed milk (Biofroxx, Guangzhou, China) for 1 h at room temperature. The membranes were then incubated with the primary antibodies (TNFα, IL-1β, CRHR1, CRHR2, GAPDH, p-PI3K, PI3K, p-AKT, AKT, p-mTOR, mTOR, and ZO-1; 1:1000 dilution, Proteintech, Wuhan, Chian) at 4 °C overnight, followed by an incubation with HRP-conjugated secondary antibody (Proteintech, Wuhan, China) for 1 h at room temperature. Signals were detected using the ECL Western blotting substrate (Millipore, Darmstadt, Germany) and quantified with imageJ software (ImageJ 1.46r, National Institutes of Health, Bethesda, MD, USA). The data were obtained from three independent experiments.