Figure 6

Cby homodimerization is dispensable for its interaction with β-catenin and for repression of β-catenin signaling activity. (A) Binding of Cby point mutants to β-catenin was evaluated by in vitro pull-down assays. Bacterially produced MBP or individual MBP-Cby protein was incubated with His-tagged β-catenin C-terminal domain (His-βcatR10-C). The protein complexes were then pulled down with amylose resin and subjected to Western blotting using anti-β-catenin antibody (top panel). The input lane was loaded with one-fiftieth of the amount of His-βcatR10-C used in the binding reactions (lane 1). One-thirtieth of each pull-down sample was run on a separate SDS-PAGE and immunoblotted with anti-MBP antibody, showing that similar amounts of MBP-Cby protein were pulled down (bottom panel). (B) The ability of Cby mutants to repress β-catenin signaling was tested by TOPFLASH assays. HEK293T cells were transfected with 60 ng of TOPFLASH or mutant FOPFLASH luciferase reporter, with or without 40 ng of an expression vector for stabilized β-catenin (β-catenin-Myc), and the indicated amounts of a Flag-tagged Cby expression vector. Luciferase activity was measured 24 hr post-transfection, and normalized to Renila luciferase activity used as an internal control. Transfections were carried out in triplicate and the means ± SD are shown. Note that, to compensate protein levels, higher amounts of plasmid DNA for the Cby mutants were used for transfection.