Tcf1 tcf7

Ten-μg protein samples were run in 10% SDS-PAGE gel and transferred onto a polyvinylidene fluoride (PVDF) membrane using the Bio-Rad Mini-Protean system (Bio-Rad Laboratories, Inc, Hercules, CA, USA). Non-specific binding was blocked by incubating the membranes in 5% skimmed milk in tris-buffered saline with Tween 20 (TBST) for 1 h and then overnight at 4 °C with the antibodies against total β-catenin (1:1000, Cell Signaling Technology, Danvers, MA, USA), free β-catenin (1:1000, Cell Signaling Technology), p-GSK-3β (1:1000, Cell Signaling Technology), GSK-3β (1: 1000, Cell Signaling Technology), TCF1/TCF7 (1:1000, Cell Signaling Technology), LEF1 (1:1000, Cell Signaling Technology), p-Stat3 (1:1000, Santa Cruz, California, USA), Stat3 (1:1000, Santa Cruz), KLF4 (1:1000, Santa Cruz), c-Myc (1:1000, Santa Cruz), vimentin (1:1000, Cell Signaling Technology), Slug (1:1000, Cell Signaling Technology), and β-actin (1:500, Santa Cruz). After overnight probing with primary antibody, the membranes were incubated with horseradish peroxidise (HRP)-linked secondary antibodies for 1 h, then washed with PBS thrice. The protein band signals were detected and developed using an enhanced chemiluminescence (ECL) detection system (Thermo Fisher Scientific Inc, Waltham, MA, USA). Protein bands were quantified using ImageJ software.

Cell and tissue lysates were lysed using RIPA lysis buffer (Beyotime, China) supplemented with the Halt™ Protease and phosphatase inhibitor cocktail (Thermo, USA). Samples were separated by electrophoresis on 8% SDS–PAGE gels and transferred onto PVDF membranes (Millipore, USA). The membranes were blocked with 5% non-fat milk, followed by incubation with primary antibodies targeting the following proteins, according to the manufacturer’s instructions: TCF1/TCF7, H3, and Actin (Cell Signaling Technology, China). Membranes were then incubated with the appropriate secondary antibodies (Cell Signaling Technology) according to the manufacturer’s instructions. Protein bands were developed and analyzed using an automatic chemiluminescence system (Tanon, China), and the intensity of the bands (pixels/band) was determined using ImageJ software as arbitrary optical density units.

Total proteins were extracted from specimens and cells using radioimmunoprecipitation assay lysis buffer with protease inhibitor on ice and quantified by bicinchoninic acid protein assay kit (Thermo, USA). The western blot was performed according to standard procedures. The following specific antibodies were applied: ALKBH5 (Millipore Corporation, USA), SOX2, Wnt5a, β-catenin, C-myc, CylinD1, LEF1, TCF1/TCF7, Met/pro-Met, Caspase-3, Caspase-7, Caspase-8, Caspase-9, MDR1, BCRP1, MRP1, and β-actin (Cell Signaling Technology, USA). The antibody information is listed in Supplementary Table S4. The membranes were incubated with HRP-labeled goat-anti-rabbit or goat-anti-mouse secondary antibodies (Cell Signaling Technology, USA). The proteins were detected and visualized by chemiluminescence (Biosciences, Foster City, CA, USA). The protein expression was analyzed by the Image J software, and β -actin was used as loading control.