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. Author manuscript; available in PMC: 2010 Feb 1.
Published in final edited form as: Mol Cancer Ther. 2009 Jan 27;8(2):458–468. doi: 10.1158/1535-7163.MCT-08-0885

Figure 4.

Figure 4

Ectopic expression of WIF1 or DN-LEF1 in TSU-PR1 cells inhibits TCF4 and β-catenin binding to SKP2 promoter sequences. A, real-time RT-PCR analysis of p21, p27, and SKP2 mRNA of stable WIF1 or DN-LEF1 transfectants in TSU-PR1 cells as mentioned in Materials and Methods. β-Actin was determined as a loadingcontrol. B, partial nucleotide sequence of human SKP2 gene promoter. The transcriptional start site (+1) and the potential TCF4/LEF1, GATA, nuclear factor-κB, AP-1, GABP, and E2F binding sites are underlined. C, chromatin immunoprecipitation assay as described in Materials and Methods. Chromatin immunoprecipitation using anti-TCF4 and anti-β-catenin antibodies was done in TSU-PR1 cells. Eluted DNA fragments were purified and used for PCR usingprimers specific for the SKP2 proximal promoter. TCF/LEF bindingelements are composed of a highly conserved consensus sequence 5′-(A/T)(A/T)CAA(A/T)G-3′. Input, 1:20 reversal cross-link chromatin DNA; NG, negative control without antibody. D, chromatin immunoprecipitation/real-time PCR assay as described in Materials and Methods. The chromatin immunoprecipitation results obtained by three independent replicate experiments are represented as percentage of input. Bars, SE. Black and white bars, chromatin immunoprecipitation signals; grid bars, signals from the no antibody control (NoAb).