Sds sample buffer

Total proteins of cell samples were extracted with lysis buffer and then quantified using the BCA method (KeyGen Biotech, Jiangsu, China). The lysate was diluted in SDS sample buffer (KeyGen Biotech) for SDS-polyacrylamide gelelectrophoresis (PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes (Roche Applied Sciences,USA). The membranes were immunoblotted at 4 °C overnight with anti-PRDX2 (Proteintech, USA), anti-c-Myc(Abcam, UK), anti-p-c-Myc(S62) (Abcam), anti-p-c-Myc(T58)(Abcam), anti-E-cadherin (Proteintech), anti-Vimentin (Proteintech), anti-N-cadherin (Proteintech), anti-GSK3β (Abcam), anti-p-GSK3β (Ser9) (Abcam), anti-AKT(1/2) (Abcam), anti-AKT1 (Abcam), anti-AKT2 (Abcam), anti-p-AKT2 (Ser474) (Abcam) and anti-GAPDH (Proteintech) antibodies at appropriate dilution concentration, followed by incubation using the appropriate second antibodies for 2 h. The bands were exposed using Pierce ECL Western Blotting Substrate (Thermo Scientific). Image J software was used to analyze the grey value of the interest protein.

Cells were cultured at a density of 1.5 × 10⁵ cells/well on 8 mm coverslips in 12-well plates. After 48 hours, coverslips were fixed by ice-cold methanol, and incubated with primary E-cadherin (Abcam), Vimentin and β-catenin (Epitomics) antibodies prior to florescent-labeled secondary antibodies. Nuclear DNA was stained with 4′, 6-diamidino-2-phenylindole (DAPI) and coverslips were mounted with FluorSave reagent (CALBIOCHEM). Immunofluorescence images were taken by Olympus inverted fluorescence microscope and were outputted by PV10-ASW 1.7 viewer software. Immunoblotting was performed as follows: Proteins were extracted with lysis buffer and then quantified by the BCA method (KeyGen Biotech). Lysates were diluted in SDS sample buffer (KeyGen Biotech) prior to SDS-PAGE, and then transferred to a polyvinylidene difluoride membrane (Roche Applied Sciences). Membranes were immunoblotted overnight at 4°C with anti-SOX17 (Millipore), anti-CyclinD1 (Abclonal), anti-C-myc, anti-DKK1 (Cell Signaling Technology), anti-E-cadherin (Abcam), anti-SOX2, anti-β-catenin, anti-Vimentin and anti-Slug and anti-N-cadherin antibodies (Epitomics), followed by the appropriate second antibodies. The bands were exposed using Pierce ECL Western Blotting Substrate (Thermo Scientific). Gel densitometry (Bio-Rad) was used to quantify immunoblot signals on exposed film.

The intestinal tissues and Caco-2 cells were homogenized and lysed using radioimmunoprecipitation assay (RIPA) lysis buffer (keygen BioTECH, Nanjing, China), supplemented with 1% phenylmethylsulfonyl fluoride (PMSF) (keygen BioTECH). The concentration of the extracted proteins was determined using the Bicinchoninic acid assay kit (keygen BioTECH). Protein solutions were mixed with sodium dodecyl sulfate (SDS) sample buffer (keygen BioTECH) in a 4:1 ratio and denatured in boiling water for 10 min. Protein samples were separated on a 10% polyacrylamide gel and then transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore). The membrane was blocked in rapid blocking solution at room temperature for 10 min, followed by overnight incubation at 4 °C with antibodies against hydroxycarboxylic acid receptor 2 (Hcar2) (Affinity Biosciences, Jiangsu, China), claudin3 (Affinity Biosciences), claudin4 (ZENBIO Biotechnology, Chengdu, China), β-actin (ABclonal, Wuhan, China), and β-Tubulin (ZENBIO Biotechnology). Subsequently, the membrane was incubated with a labeled secondary anti-rabbit antibody for 1 h, and the immunoreactive protein bands were detected using an enhanced chemiluminescence (ECL) kit (Millipore) and visualized using the Bio-Rad ChemiDoc™ Touch Imaging System (Bio-Rad, Hercules, CA, USA). Image analysis was performed using Image Lab and ImageJ software.