Figure 5. Protein–DNA complex formation between β-catenin/TCF4 and the corresponding binding domain of the GLCE promoter.

γ-³²P-labelled oligonucleotides of the sequence corresponding to the putative β-catenin–TCF4 binding site of the GLCE promoter were incubated with a nuclear extract from SW480 cells, and the DNA–protein complex formed was analysed on 6% non-denaturing polyacrylamide gel. After electrophoresis, bands were visualized by autoradiography. For the competition binding, 30-fold excess of unlabelled oligonucleotides were included in the incubation mixture (lane 2). Two shifted bands corresponding respectively to DNA–TCF4 (lower band) and DNA:TCF4/β-catenin (upper band) complexes were detected [29]. For the supershift analysis, 0.5 μg of an irrelevant monoclonal antibody (anti-β-integrin, lane 3) or anti β-catenin antibody (lane 4) were added.