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. 2008 Mar 24;180(6):1087–1100. doi: 10.1083/jcb.200710050

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Copyright © 2008, The Rockefeller University Press

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Figure 5. — c-Jun mediates Dvl association with the functional TCF–β-catenin complex. (A) c-Jun associates with the c-myc promoter in a TCFs-dependent and Wnt-induced manner. The ChIP assays were performed in HEK293T cells as indicated. To test whether the TCF-binding site is required for the binding of Dvl and c-Jun to the promoter, 100 ng of pBV-TBE1/2-luc (the wild type of the c-myc promoter) or pBV-TBE1m/2m-luc (a TCF binding site mutant of the c-myc promoter) plasmids (He et al., 1998) were transfected in HEK293T cells in a 100-mm plate. Before ChIP assays, the cells were stimulated with Wnt-3a CM for 3 h. The primer pairs used to detect exogenous c-myc promoter were derived from a pBV vector sequence: 5′-AGGTACGGGAGGTACTT-3′ and 5′-ACCAACAGTACCGGAAT-3′. (B) c-Jun depletion suppresses the binding of Dvl-3 to the c-myc promoter. HEK293T cells were transfected with c-Jun siRNA for 72 h and then treated with Wnt-3a CM for 3 h. The ChIP assays were performed as indicated. (C) c-Jun depletion disrupts the nuclear Dvl/TCF-4 association. HEK293T cells were treated with c-Jun siRNA for 72 h and then stimulated by Wnt-3a CM for 3 h. The nuclear extracts were immunoprecipitated by an anti–Dvl-3 antibody and then detected by an anti–TCF-4 antibody. (D) TCF-Dvl functions as well as TCF-4 in mediating Wnt-3a–stimulated transcription. HEK293T cells in 24-well plates were transfected with 20 ng of LEF-luc plasmid and either 5 ng of TCF-4 or 37.5 ng of TCF-Dvl. Western analysis of TCF-4 and TCF-Dvl expression is shown. Error bars indicate SD of duplicate data in one experiment; the results were repeated three times. (E) TCF-Dvl can relieve the suppression of c-myc expression caused by c-Jun siRNA. HEK293T cells in a 24-well plate were transfected with 0.5 μg of c-Jun siRNA or control siRNA. 5 ng TCF-4 or 20 ng TCF-Dvl was used to rescue the c-Jun depletion. Error bars indicate SD of three independent experiments. (F) TCF-Dvl can relieve the suppression of ventral marker expression caused by c-Jun MO in zebrafish. 8 ng of c-Jun MO were injected with 0.5 ng of GFP, 0.1 ng of TCF-Dvl, 0.5 ng of TCF-Dvl, or 0.15 ng TCF-4 mRNA, respectively, into one-cell embryos. Graph shows the penetrance of rescue.