Fig. 5

Nkd1 interacts with β-catenin.

To determine if Nkd1 and β-catenin interact, wnt8 +/- nkd1^myc or nkd1^G2A-myc was injected at the 1 cell stage and harvested at dome stage for immunoprecipitation with anti-β-catenin (A). Each lane represents the equivalent of 17.5 embryos. Note, this experiment was part of a larger experiment involving the co-immunoprecipitations shown in Fig. 2 (B) and as such share some of the same controls. (B) nkd1^myc +/- wnt8 was injected into 1 cell stage embyros and harvested at dome stage, immunoprecipitated with anti-myc and western blotted with anti-β-catenin. The interaction between Nkd1 and β-catenin is much stronger in the presence of exongenous Wnt. Slight non-specific binding of the anti-myc antibody is observed in the control lane. A no primary antibody control showed no banding pattern (not shown). C) nkd1^myc was co-injected with increasing doses of dsh^HA (0pg, 125pg and 250pg) harvested at dome stage, immunoprecipitated with anti-myc and western blotted with anti-β-catenin. The astericks identify non-specific banding patterns. D) The interaction of luciferase-tagged β-catenin (β-catenin-Lux) with wild type Nkd1 and Nkd2 (left panel) and Nkd1 mutants (right panel) was tested using the LUMIER assay in HEK293T cells. Numbers above Wild Type Nkd1 refer to amino acid positions in human Nkd1. Axin data has been previously reported [28] and is shown here for completeness. Data are shown as the mean of 2 samples +/- standard deviation (left panel) and interaction intensities of less than 50% (-), 50–75% (+) and >75% (++) of wild type are indicated (right panel). clarity we refer to NHR1 as Naked Homology Region 1 (116–192aa), which encompasses the EF hand (143–154aa). The NHR1 region has also been referred to as NH2 [56] as well as EFX [10].