Figure 7.
Nkd1 cooperates with Axin1 to inhibit the Wnt pathway. (A) Nkd1 interacts with Axin. HEK293T cells were transfected with luciferase-tagged Nkd1 (Nkd1-FF-Luc) and 3XFlag-tagged Axin1 or Axin2. Interactions were detected by luciferase assay following anti-flag immunoprecipitation. Data are shown as the mean of two samples ±standard deviation. (B) Endogenous Axin1 interacts with 3XFlag-Nkd1. HEK293T cells were transfected with Flag-tagged Nkd1 or empty vector. Interactions were detected by anti-Flag immunoprecipitation followed by immunoblotting. The migration of endogenous Axin was confirmed in total cell lysates by comparison with transiently transfected Axin1 (left two lanes). * Indicates a nonspecific band. (C) Schematic of Nkd1 deletion mutants and the amino acid sequence of the His-rich carboxy-terminus is shown. (D) Axin1 interacts with the C-terminal His-rich tail of Nkd1. HEK293T cells were transfected with luciferase-tagged Axin1 (Axin1-FF-Luc) and the indicated Flag-Nkd1 constructs. Cell lysates were subject to anti-Flag immunoprecipitation and the presence of Axin1 was assessed by luciferase assay. Data are expressed as the mean of two samples±range. (E) Multiple regions of Nkd1 are required for efficient inhibition of TOPflash activity. HEK293T cells were transfected with TOPflash and the indicated Flag-Nkd1 constructs. Cells were treated overnight with or without Wnt3A and TOPflash activity was measured by luciferase assay. Data are shown as the mean of two samples ±standard deviation.