Tgf β

Using a hot air oven, tissue section slides were heated at 60 °C for about 25 minutes (Venticell, MMM, Einrichtungen, Germany). The tissue sections were de-paraffinized in xylene and rehydrated with a graded alcohol series. The sample slides were boiled in 10 mM sodium citrate buffer in a microwave for antigen retrieval. Next, 0.03% hydrogen peroxide containing sodium azide was applied for 5 minutes to block endogenous peroxidase. Tissue sections were then washed gently with wash buffer, then incubated with biotinylated primary antibodies against Hsp70 (1:500), Bax (1:200) and TGF-β (1:500) (Abcam, UK) for 15 minutes. The tissue sections were washed gently with wash buffer and placed in a buffer bath. Next, the slides were placed in a humidified chamber with a sufficient amount of streptavidin-HRP (streptavidin conjugated to horseradish peroxidase in PBS containing an anti-microbial agent) for 15 minutes. Diaminobenzidine-substrate-chromagen was added to the tissue sections and incubated for another 5 minutes. This was followed by washing and counterstaining with hematoxylin for 5 sec. The tissue sections were then dipped in weak ammonia (0.037 mol/L) 10 times and washed with distilled water. Positive immunohistochemical findings were observed by the formation of brown staining and assessed by light microscopy.

Protein lysates of Vector‐TEVs, Foe‐TEVs or Foe‐Th cells were prepared according to standard procedures, and the contents of proteins were assessed by the BCA protein concentration measurement kit (Thermo Fisher Scientific). Subsequently, the samples were isolated using Bis‐Tris gel (Invitrogen), and then transferred to polyvinylidene difluoride membranes (Millipore). The membrane was blocked in TBS‐T in 5% BSA for 1 h, incubated with primary antibodies overnight at 4 °C, then rinsing the membranes with PBS. Then, the membranes were covered with secondary antibody. Incubated at room temperature for 2 h, signals were assessed by enhanced Chemiluminescence Advanced System (GE Healthcare). The primary antibodies involving against CD9 (1:1000, Abcam), CD63 (1:1000, Abcam), Tubulin (1:1000, Abcam), TSG101(1:1000, Abcam), CTLA‐4 (1:1000, Abcam), TGF‐β (1:1000, Abcam), IL‐10 (1:1000, Abcam), PDCD‐1 (1:1000, Abcam), CD35 (1:1000, Abcam), CD73 (1:1000, Abcam), LAG3 (1:1000, Abcam), TIGIT (1:1000, Abcam), calnexin (1:1000, Abcam), histone 3 (1:1000, Abcam), and GM13 (1:1000, Abcam), ITGA4 (1:800, Abcam), ITGAL (1:800, Abcam), ITGB1 (1:800, Abcam), ITGB2 (1:800, Abcam) and secondary antibody Alexa Fluor Plus 800 (1:10 000, Thermos) were used.

The tissue was harvested at indicated time points and lysed in a lysis buffer (0.5% Nonidet P-40, 10 mM Tris, pH 7.4, 150 mM Nacl, 1 mM EDTA, 1 mM Na₃VO₄) with a protease inhibitor (1 mM PMSF). BCA assay was used to quantify protein level. Cell lysates (30 μg protein) were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Immobilon; Millipore, Bedford, MA). For immunoblotting, primary antibodies against α-SMA, TGF-β, α-tubulin and β-actin (1:1000) (Abcam, Cambridge, UK) and IgG-HRP secondary antibody (1:2000) were used. Chemiluminsescent signals were acquired using Storm 860 PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Densitometric analysis was performed using Image J 5.0 software. Four biological replicates were performed.

The protein was extracted from GC cells and tissues and transferred to polyvinylidene difluoride (PVDF) membranes. The transferred membranes were blocked in 5% non-fat powdered milk for 2h and incubated with primary antibodies overnight at 4°C. The membranes were then incubated for 2h in secondary antibodies at room temperature. The primary antibodies used in this study were as follows: Smad3 (diluted 1:500, Abcam), TGF-β, c-Myc, CDK2, CDK4, CDK6, GAPDH (diluted 1:1000, Cell Signaling Technology). GAPDH was used as an internal control.