Epha2

Total protein of colonic tissues or Caco2 cells were extracted via radioimmunoprecipitation assay (RIPA) buffer containing phosphatase and protease inhibitor cocktail. For immunoblotting analysis, lysates were separated on 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels with constant voltage (100 V, 90 minutes), and transferred to polyvinylidene fluoride (PVDF) membranes with constant current (300 mA, 90 minutes) in ice-water baths. Membranes were subsequently blocked in 5% (weight/volume) bovine serum albumine, incubated with specific primary antibodies (occludin, claudin-1, ephrinA1, or ephA2; Abcam, Cambridge, MA, USA) and horseradish peroxidase-conjugated second antibodies, finally the protein bands were developed by a Super Signal West Pico Substrate (Pierce, Rockford, IL, USA) and quantified on the Fluor Chem FC3 system (ProteinSimple, San Jose, CA, USA). For immunoprecipitation assays, protein lysates were pre-cleared with agarose-protein A/G (Beyotime, Beijing, China), and then incubated with anti-phosphotyrosine antibody, clone 4G10 (pre-incubated with agrose-protein A/G), centrifugated and the deposition was collected (immunoprecipitating complex). Subsequently, the complex was used for electrophoretic analysis by immunoblotting with anti-ephA2 antibodies as described above.

For paraffin section (fixed with 4% paraformaldehyde), routine dewaxing and hydration and then use for staining was performed. For the Caco2 monolayer cell slide, fixation in 4% paraformaldehyde for 15 minutes was used for staining. Firstly, the paraffin sections or cell slides were blocked with 10% donkey serum for 1 hour at room temperature. Next, the preparations were incubated with primary antibodies (occludin, claudin-1, ephrinA1, or ephA2; Abcam) overnight at 4°C, fully washed and combined with Alexa Fluor 488 secondary antibodies (Invitrogen, Carlsbad, CA, USA) for 60 minutes at room temperature. Finally, the nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI, 1 μg/mL; Beyotime). The fluorescent imaging was viewed on a confocal laser scanning microscope (Nikon, Tokyo, Japan), and analyzed using NIS Elements Viewer Software (Nikon).

Harvested cells (U87 and U251) were lysed using RIPA (Beyotime Institute of Biotechnology) buffer on ice for 30 min and were centrifuged at 17,000 × g for 45 min at 4°C. The protein concentrations were measured by the BCA protein assay kit (Beyotime Institute of Biotechnology, Jiangsu, China). The proteins were processed by SDS-PAGE electrophoretically transferring to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked by Tween-Tris-buffered saline (TTBS) containing 5% non-fat milk for 2 h at room temperature and then incubated with primary antibodies as follows: USF1 (Santa Cruz Biotechnology), ALDH1A1 (Proteintech, USA), MMP-14 (Proteintech, USA), MMP-2 (Proteintech, USA), VE-cadherin (Abcam, UK), EphA2 (Abcam, UK), ERK (Abcam, UK), p-ERK (Abcam, UK), and GAPDH (Proteintech, USA) overnight at 4°C. After washing three times with TTBS, membranes were incubated with horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature and then developed with enhanced chemiluminescence (ECL) kit (Santa Cruz Biotechnology) and scanned by ChemImager 5500 V2.03 software according to the manufacturer’s protocol. The relative integrated density values (IDVs) were calculated using Fluor Chen 2.0 software based on GAPDH as an internal control.