Foxp3

Liver sections from mice were crushed in liquid nitrogen and then lysed in NP 40 lysis buffer (50 mM Tris-Cl, pH 8.0, 100 mM NaCl, 5 mM MgCl₂, 0.5% (v/v) Nonidet P-40, protease inhibitor cocktail, Beyotime Biotech, Beijing, China) on ice for 30 min. The protein concentration was determined using a BCA Protein Assay Kit (Beyotime Biotech, Beijing, China) following the manufacturer’s procedure. Samples were run on a 10% SDS polyacrylamide gel, transferred to a PVDF membrane, and immunoblotted with primary antibodies Foxp3 (1:2000 dilutions, proteintech, Chicago, USA), RORγt (1:2000 dilutions, proteintech, Chicago, USA). After incubation with secondary antibodies (1:2000, Sigma, San Diego, CA, USA), the signal was detected using the Immun-Star Western Chemiluminescence kit (BioRad, Hercules, CA, USA), quantified using the ChemiDoc XRS Imager and analyzed with the software Image Lab from Bio-Rad (BioRrad, Hercules, CA, USA). Tublin was used as endogenous control and the data were normalized by Tublin levels.

The HCT-116 cell line was purchased from ATCC, USA. B. infantis CGMCC No. 0313-2 (Batch No. 2017012) was provided Kexing Biological Products Co., Ltd, Shandong, China. Anti-PD-L1 was purchased from BIOX cell, New Hampshire, USA. Protein marker (m000624) was purchased from Kingsley Biotechnology Co., Ltd, Nanjing, Jiangsu, China. Polyvinylidene fluoride (PVDF) membranes were purchased from the GE company, Fairfield, Connecticut, USA. GAPDH antibody, phospho-PI3K antibody, and phospho-mTOR antibody were purchased from Abcam, USA. PD-L1, PD-1, Foxp3, AKT1, and mTOR antibodies were purchased from Proteintech, USA. PI3K, phospho-Akt, PTEN, and phospho-PTEN antibodies were purchased from CST, USA. Sensitive chemiluminescence solution was sourced from Millipore, USA. PrimeScript RT reagent kit with gDNA Eraser was purchased from TaKaRa Bio, Japan. The qRT-PCR kit SYBR Premix Ex Taq II (TLI RNaseH Plus) was from TaKaRa. TPY agar medium and TPY broth were purchased from Qingdao Haibo Biotechnology Co., Ltd, China. All primers were purchased from China Bio Engineering Co., Ltd. The turbidimetric tube was purchased from China Wenzhou Kangtai Biotechnology Co., Ltd.