Primary antibody against occludin

Tissues were lysed in RIPA buffer with the addition of protease and phosphatase inhibitor cocktail (200 mM AEBSF, 30 μM Aprotinin, 13 mM Bestatin, 1.4 mM E64, 1 mM Leupeptin in DMSO, and 0.1 M EDTA). The lysate was centrifuged at 12,000 g at 4°C for 30 min and the protein concentration was measured using a BCA protein quantitative reagent kit (Beyotime, Shanghai, China). An equal amount of protein from each sample was separated by 8% SDS-PAGE and transferred to a PVDF membrane (Millipore, MA, United States). After blocking the membrane with 5% skimmed milk in tris buffered saline with tween-20 (TBST), the membrane was incubated with primary antibody against occludin, ZO-1, and TLR4 (Proteintech Group Inc., IL, United States) overnight at 4°C. GAPDH was used as a control. The membranes were then washed three times with TBST before being probed with the horseradish peroxidase-conjugated secondary antibodies. Signals were detected using a chemiluminescence system (ECL; Bio-rad, CA, United States; Tanon 4600, China) and the intensities were quantified using the ImageJ software.

HWH was supplied and analyzed by Zhen Jiu Co., Ltd. (Dalian, China). Cyclophosphamide was purchased from Aladdin (Shanghai, China). Levamisole hydrochloride was purchased from Shanxi Hongbao Veterinary Pharmaceutical Co., Ltd. (Yuncheng, China). The First Strand cDNA Synthesis Kit and the SYBR PrimeScript^™ RT-PCR Kit were purchased from Takara Bio Co., Ltd. (Shiga, Japan). The primer sequences of TNF-α, IL-6, and IL-1β were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The primary antibody against occludin was purchased from Proteintech Group Co., Ltd. (Wuhan, China). Antibodies against claudin and ZO-1 were from Wanleibio Co., Ltd. (Shenyang, China). E.Z.N.A.^® Stool DNA Kit was purchased from Omega Bio-Tek Co., Ltd. (Norcross, GA, USA). AxyPrep DNA Gel Extraction Kit was purchased from Axygen, Inc. (New York, NY, USA).