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Figure 1 | Journal of Molecular Signaling

Figure 1

From: Chibby suppresses growth of human SW480 colon adenocarcinoma cells through inhibition of β-catenin signaling

Figure 1

Forced expression of Cby in SW480 colon cancer cells promotes translocation of nuclear β-catenin towards the cytoplasm, resulting in inhibition of endogenous β-catenin signaling. (A) SW480 cells were transiently transfected with a control empty vector or an expression plasmid for Flag-tagged CbyWT or Cby∆NLS, and doubly immunostained with anti-Cby (red) and anti-β-catenin (green) antibodies. Nuclei were stained with DAPI. A merged image of β-catenin and DAPI is also shown. (B) Quantitative analysis of the results in (A). The subcellular localization of endogenous β- catenin was scored as follows: N>C, predominantly nuclear; N=C, evenly distributed between the nucleus and cytoplasm; N<C, predominantly cytoplasmic. Error bars represent the means ± SD of three independent experiments. For cells transfected with Cby plasmids, β-catenin localization was scored only in those expressing ectopic Cby. (C) Western blot analysis of Cby expression in SW480 cells using anti-Cby antibody. Note that, to compensate protein levels, higher amounts of DNA for Cby∆NLS were used for transfection. The anti-Cby antibody detected both exogenous and endogenous proteins. An asterisk indicates a non-specific band that overlaps with Flag-Cby. GAPDH was used to confirm equal loading. (D) The ability of Cby to repress endogenous β- catenin signaling was tested by TopFlash assays. SW480 cells were transfected with 100 ng of TopFlash luciferase reporter and the indicated amounts of a Flag-tagged Cby expression plasmid. Luciferase activity was measured 24 h post-transfection, and normalized to Renilla luciferase activity used as an internal control. All transfections were carried out in triplicates and the means ± SD are shown.

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