Fig. (6).

Simultaneous stimulation of both canonical and non-canonical Wnt signaling leads to differential coactivator utilization by nuclear Iý-catenin leading to preferential interaction between Iý-catenin and p300. To examine the interaction of nuclear Iý-catenin with the coactivators CBP or p300 after Wnt stimulation, wild type (WT) 3T3 cells were treated with Wnt3a, Wnt5a, or Wnt3a+Wnt5a conditioned media (CM) versus control for 24 h, after which nuclear lysates were prepared and subjected to immunoprecipitation (IP) with CBP, p300, or control antibody, followed by immunoblot for Iý-catenin. Wnt3a-only conditioned media markedly increased the coimmunoprecipitation of Iý-catenin with both CBP and p300 compared to control treated cells (compare lanes 1 and 2). Whereas Wnt5a-only conditioned media caused no increase in the amount of Iý-catenin associated with p300, a marked decrease in the amount of Iý-catenin associated with CBP compared to control (compare lanes 1 and 3) was observed. Co-treatment with Wnt3a and Wnt5a conditioned media caused a decrease in the amount of Iý-catenin associated with CBP compared to treatment with Wnt3a alone, while a conspicuous increase in p300 associated Iý-catenin was observed under these conditions. Ab, antibody.