Anti tcf4

EMSA experiment was conducted as depicted previously.²⁵ The nuclear extracts were obtained from AGS cells. Probes were produced by annealing single‐strand oligonucleotides containing the TCF4 consensus sequence of circ_SMAD4 promoter and labelling the ends with [γ‐32P] ATP using T4 polynucleotide kinase (TaKaRa Bio). Anti‐TCF4 (Proteintech) and anti‐IgG (Santa Cruz) were used as primary antibodies.

Cultured DPCs were lysed with RIPA buffer (Beyotime, Shanghai, China), the protein concentration was measured with BCA, and a small part of the lysate was prepared as input. The rest of the lysate was incubated with immunoglobulin G (IgG; negative control), anti-Twist1 (Santa Cruz, CA, United States), or anti-Tcf4 (Proteintech, Shanghai, China) overnight. Then protein A/G agarose was added to couple to the antibody. Finally, the immunoprecipitate was eluted and analyzed by Western blot.

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.