Association of Cby with 14-3-3 influences β-catenin signaling. (A) Effects of different 14-3-3 isoforms on β-catenin–mediated transcriptional activation were evaluated by TopFlash assays. (B) The ability of Cby mutants to repress β-catenin signaling was tested by TopFlash assays. HEK293T cells were transfected with 10 ng of TopFlash or mutant FopFlash luciferase reporter with or without 10 ng of an expression vector for stabilized β-catenin (β-catenin–Myc), 200 ng of a HA-tagged 14-3-3 plasmid, and the indicated amounts of a Flag-tagged Cby expression vector. Luciferase activity was measured 24 h after transfection and normalized to Renila luciferase activity used as an internal control. Transfections were performed in triplicate and the means ± SD are shown. Western blot analysis with anti-Cby antibodies showed that Cby proteins were expressed at similar levels. Note that, to compensate protein levels, higher amounts of DNA for CbyS18A/20A, CbyS20A, and CbyΔ1-22 were used for transfection. White lines indicate that intervening lanes have been spliced out.