P jnk

The proteins were extracted using total protein extraction kit (CWBio). Proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to PVDF membrane. Primary antibody directly against PARP, Fas, Fasl, casepaes-3 and GAPDH were from Cell Signaling Technology, Inc. (Danvers, MA, USA). The primary antibodies against p-ERK1/2, p-JNK, p-P38 were puchased from Sigma-Aldrich; Merck KGaA, while p-ERK5, ERK5, ERK1/2, JNK, P38MAPK were from Cell Signaling Technology, Inc. HRP-conjugated secondary antibodies are from CWBio. And the signals were visualized with BeyoECL Plus (EMD Millipore, Billerica, MA, USA).

IEC-6 cells were plated into 6-well plates at a density of 3 × 10⁵ cells/mL. When cells reached 60% confluence, 10 ng/mL TGF-β1 and TFA (0, 5, 10 and 15 μg/mL) were added to the plates and incubated for 48 h. Each concentration group was performed in triplicate. Protein lysates were prepared using RIPA lysis buffer. Lysates were then subjected to SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes. After blocking with non-fat milk, blots were incubated overnight at 4 °C with the indicated antibodies. After incubation for 24 h, membranes were washed and incubated for 2 h at room temperature with corresponding secondary antibodies. For protein detection, membranes were developed with SuperSignal west femto maximum sensitivity substrate (Pierce, Rockford, IL, United States) and the Gel-Pro Analyzer 6.0 software was applied for image analysis. E-cadherin (1:500), ZO-1 (1:500), Vimentin (1:500), N-cadherin (1:500), p-ERK (1:500), ERK (1:500), p-p38 (1:500), p38 (1:500), p-JNK (1:500), JNK (1:500) and GAPDH (1:1000) antibodies were purchased from Sigma-Aldrich, and anti-mouse secondary antibodies were obtained from Proteintech (Chicago, IL, United States).