Proliferating cell nuclear antigen (pcna)

A375 and A875 cells were harvested with RIPA buffer (Thermo Fisher Scientific) in compliance with the manufacturer’s instructions. Then, the protein concentration was examined using BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amount protein samples were loaded on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes. Subsequently, membranes were blocked with 1% bovine serum albumin for 1 h at room temperature and probed with primary antibodies at 4°C overnight, and GAPDH (1:2000 dilution; Abcam, Cambridge, MA, USA) was used for normalization. After extensive washing, the membranes were incubated with a goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:2000 dilution; Abcam) for 1 h. Finally, protein bands were visualized and analyzed with enhanced chemiluminescent method and Image J software (National Institutes of Health), respectively. The primary antibodies were listed as follow: hexokinase 2 (HK2; 1:1000 dilution; Abcam), E-cadherin (1:1000 dilution; Abcam), vimentin (1:1000 dilution; Abcam), N-cadherin (1:1000 dilution; Abcam), ITGA9 (1:1000 dilution; Abcam). ProliferatingCellNuclearAntigen (PCNA; 1:1000 dilution; Abcam), cyclin D1 (1:1000 dilution; Abcam), B-cell lymphoma-2 (Bcl-2; 1:1000 dilution; Abcam).

Sections of small intestinal and colonic tissue from all rats sacrificed for immunofluorescence staining were frozen in tissue freezing medium prior to cryo-embedding and sectioning. For immunostaining, 6 µm frozen sections were fixed in cold acetone for 15 min. Thereafter, tissues were permeabilized with 0.2% Triton X-100 in PBS for 20 min, and blocking was achieved by incubation with 5% goat serum. Incubation with primary antibodies ZO-1 (1:100; Zymed Laboratories Inc., San Francisco, CA, USA), occludin (1:200; Zymed Laboratories Inc.), heme oxygenase-1 (HO-1) (1:500; Abcam Inc., Cambridge, MA, USA) and proliferating cell nuclear antigen (PCNA) (1:500; Abcam Inc.) was performed overnight at 4°C. After three washes with PBS, the sections were incubated with Alexa 594-conjugated secondary antibodies (1:500; Molecular Probes) at room temperature for 2 h in the dark. Sections were then washed and mounted under coverslips using ProLong Gold antifade reagent with DAPI (Invitrogen, Carlsbad, CA, USA). The stained sections were visualized and photographed using a Nikon fluorescence microscope (NIS-Elements systems; Nikon Instruments Inc., Melville, NY, USA).

Total proteins were extracted using RIPA buffer (Beyotime) and quantified using BCA Kit (Beyotime). The equivalent amount of protein was isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Then, the membranes were closed with 5% non-fat milk and incubated with the primary antibodies against caspase 3 (1:1000, Beyotime), B-cell lymphoma-2 (Bcl-2; 1:2000, Abcam, Cambridge, MA, USA), Bcl-2-associated x protein (Bax; 1:5000, Abcam), E-cadherin (1:1000, Abcam), Vimentin (1:2000, Abcam), FOXP2 (1:1000, Abcam), proliferating cell nuclear antigen (PCNA; 1:5000, Abcam) or GAPDH (1:5000, Abcam) at 4°C overnight. After incubated with the secondary antibody (1:2000, Abcam) for 1 h, the membranes were treated with enhanced chemiluminescence reagent (Beyotime) to visualize the protein signals.