Claudin 3

Inulin was obtained from Yuanyebio Biotechnology Co., Ltd, (S11143, Shanghai, China). Ampicillin (Cat# A5354), neomycin (Cat# N6386), metronidazole (Cat# 16677), vancomycin (Cat# V2002) were bought from Sigma Aldrich (St. Louis, MO, USA). Specific antibodies including ZO-1 (1:1000; #AF5145; RRID: AB_2837631), Occludin (1:1000; #DF7504; RRID: AB_2841004), Claudin-3 (1:1000; #AF0129; RRID: AB_2833313) and β-actin (1:1000; #AF7018; RRID: AB_2839420) were purchased from Affinity Biosciences (OH, USA). S100A8 (#70802) and HDAC3 (#60538) antibody were bought from Cell Signaling Technology (Boston, USA). Acetylated-H3 antibody (Cat# ab47915; RRID: AB_873860) was purchased from Abcam (Cambridge, England). Tumor necrosis factor (TNF)-α (Cat# 430915) and interleukin (IL)-1β (Cat# 432615) enzyme-linked immunosorbent assay (ELISA) kits were obtained from Biolegend (San Diego, California, USA). Myeloperoxidase (MPO) (A044-1-1) assay kit was bought from Nanjing Jiancheng Bioengineering Institute (Nanjing, China,).

Deoxycholic acid (DCA), cholic acid (CA), and vancomycin were bought from Sigma Aldrich (St. Louis, MO, USA). SBI-115, MDL-12330A, and H89 were purchased from MedChemExpress (MCE, USA). The primary antibodies, including phosphorylation-p65 (p-p65, #AF2006; RRID: AB_2834435), p-65 (#AF5006; RRID: AB_2834847), p-IκB (#AF2002; RRID: AB_2834433), IκB (#AF5002; RRID: AB_2834792), Occludin (#DF7504; RRID: AB_2841004), ZO-1 (#AF5145; RRID: AB_2837631), Claudin-3 (#AF0129; RRID: AB_2833313) and β-actin (#AF7018; RRID: AB_2839420) were obtained from Affinity Biosciences (OH, USA). NLRP3 (#15101), ASC (#67824), and IL-1β (#12242) were bought from Cell Signaling Technology (CST, Boston, USA). Goat anti-rabbit or Rabbit anti-mouse secondary antibodies were bought from ImmunoWay Biotechnology Company. Mouse TNF-α (Cat #430915) and IL-1β (Cat #432615) ELISA assay kits were obtained from Biolegend (CA, USA). Myeloperoxidase (MPO) assay kit was bought from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Tissue protein extraction reagent (78510, Thermo Fisher Scientific) was added to tissues or cells to extract the total proteins, and the protein content was determined using a BCA Protein Assay Kit (23225, Thermo Fisher Scientific, California, USA). The protein samples were separated by 8%, 12%, and 15% SDS‒PAGE and then transferred to PVDF (IPVH00010, Thermo Fisher Scientific) membranes using the wet transfer method. After blocking at room temperature for 3 h with 5% skimmed milk powder, specific primary antibodies were added and samples were incubated at 4 °C overnight. After washing with TBST the next day, blots were incubated with murine or rabbit secondary antibodies for 2 h at room temperature. Finally, the membranes were washed again with TBST, and a Western blot detection system (Tanon 4500, Shanghai, China) was used to measure protein expression. The specific primary antibodies used in this experiment included β-actin (4967S), AMPK (2532S), p-AMPK (2535S), mTOR (2972S), p-mTOR (2971S), p65 (8242S), p-p65 (3033S), p38 (9212S), p-p38 (9211S), p62 (5114S), LC3 (2775S), GFP (ZY60501M), ZO-1 (AF5145), Occludin (DF7504), Claudin-3 (AF0129). Anti-GFP antibody were purchased from Zeye Biotechnology (Shanghai, China), ZO-1, Occludin and Claudin-3 were purchased from Affinity Biosciences, and all other antibodies were purchased from Cell Signaling Technology.

The total protein of colon and mammary gland were harvested using a tissue protein extract (Thermo Fisher Scientific, USA) as previously described [22 (link)]. 10% or 15% SDS-PAGE were used to separate proteins and then combined with 0.45 μm PVDF membranes. After being blocked in 5% skim milk, the prepared PVDF membranes were incubated at 4 ℃ overnight with specific primary antibodies, including ZO-1, Occludin, Claudin-3, p-p65, p65, p-IκB, and IκB obtained from Affinity Biosciences (OH, USA), and TLR4, NLRP3, ASC, IL-1β and β-actin obtained from Cell Signaling Technology (CST, Boston, USA). After washing three times with TBST, the PVDF membranes were treated with Goat anti-rabbit or Rabbit anti-mouse IgG (1:20,000, Affinity Biosciences, OH, USA) for 2 h at room temperature. Ultimately, the proteins were determined by the ECL plus western blotting Detection System (Tanon, China).