Anti tcf1

Pierce Co-IP Kits (Thermo Scientific) were used for the isolation of natural protein complexes. Our approach is briefly summarized in the following steps. Primary antibodies against β-catenin (0.5 μg/μL, Proteintech) or HIF-1α (0.165 μg/μL, Abcam) were first covalently immobilized to AminoLink Plus coupling resins for 2 h at room temperature in a tube rotator, with IgG serving as the negative control. Cells were solubilized in lysis buffer (Tris 0.025 M, NaCl 0.15 M, EDTA 0.001 M, NP-40 1%, and glycerin 5%), and 500 μL of the cell lysates incubated with pre-prepared anti-β-catenin or anti-HIF-1α primary antibody-conjugated resins overnight at 4 °C in a tube rotator. After incubation, the natural protein complexes were eluted and analyzed by Western blotting with anti-β-catenin (1:5000, Proteintech), anti-HIF-1α (1:500, Abcam), anti-TCF1 (1:1000, Proteintech), and anti-TCF4 (1:1000, Proteintech) primary antibodies as described previously.

CDX/PDX tumors and CRC tissues of patients were fixed in 4% PFA, embedded in paraffin, and sectioned. Tumor sections were dewaxed with dewaxing agent and dehydrated in graded alcohol concentrations. After antigen retrieval with heated citrate buffer and blocking with 10% goat serum, the following primary antibodies were incubated overnight at 4 °C: anti-Ki-67 (Proteintech, 1:5000), anti-HIF-1α (1:200, Abcam), anti-GLUT1 (1:500, Abcam), anti-HK2 (1:100, Abcam), anti-PKM2 (1:200, CST), anti-LDHA (1:200, Proteintech), anti-MCT4 (1:50, Santa), anti-Phospho-AKT (1:100, Abcam), anti-β-catenin (1:1000, Proteintech), and anti-TCF1 (1:200, Proteintech). Next day, the sections were incubated with HRP-conjugated anti-rabbit/mouse IgG and detected by 3,3′-diaminobenzidine (DAB) staining. The nuclei were stained with hematoxylin. The stained sections were photographed using an Axio Scope A1 microscope (Zeiss, Germany) at 200× magnification.