Anti mouse secondary antibodies

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).

The cells were washed with PBS and then lysed using RIPA buffer containing 1% protease inhibitor (both Sigma-Aldrich; Merck KGaA). Equal amounts of protein were then resolved using 10% SDS-PAGE. Protein concentrations were quantified using the Pierce protein assay kit (cat. no. UA269551, Thermo Fisher Scientific, Inc.). The separated proteins (20 µg/lane) were carefully transferred to polyvinylidene fluoride membranes at 4°C for 2 h. Subsequently the proteins were blocked using 5% skimmed milk (BD Biosciences) and probed with the following primary antibodies: Anti-ESE3 (1:1,000; cat. no. ab24337), anti-E-cadherin (1:1,000; both Abcam; cat. no. ab76055) and β-actin (1:5,000; cat. no. E4D9Z; Cell Signaling Technology, Inc.) for 2 h at room temperature. Following primary incubation, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit (1:10,000; ProteinTech Group, Inc; cat. no. SA00001-2)/anti-mouse secondary antibodies (1:10,000; ProteinTech Group, Inc; cat. no. SA00001-1) for 1 h at room temperature. Protein quantification was performed using Image Lab Software (version 5.2.1, Bio-Rad Laboratories, Inc).