Fig. 4. MAK interacts with Frizzled and Dishevelled. (A) Fz3 RNA recruits MAK to the cell membrane in animal pole cells. xMAK-GFP RNA was injected either with membrane-targeted dsRed RNA or with dsRed and Fz3 or Fz2 RNAs and GFP fluorescence was visualized in animal pole cells isolated from injected embryos at stage 10. xMAK-GFP is distributed in a punctate cytoplasmic pattern. Membrane-targeted dsRed RNA allows visualization of the cell membrane. Upon Fz3 RNA coexpression, xMAK-GFP is recruited to the cell membrane. The bottom right panel shows lack of effect of Fz2 RNA on xMAK-GFP localization (compare with top right panel). Bars on top left and top right panels are 5 µm. (B) MAK co-immunoprecipitates with Dsh. Lysates of embryos injected with Myc-MAK RNA (2 ng) and/or with HA-Dsh RNA (4 ng) into each cell of four-cell embryos were collected at stage 10 and immunoprecipitated with anti-HA antibodies. Western analysis with anti-Myc antibodies reveals a complex of Dsh and MAK. (C) In vitro phosphorylation of Dsh by MAK. Autoradiography (upper panel) reveals phosphorylation of in vitro translated Myc-Dsh that was co-immunoprecipitated with Myc-MAK or Myc-MAK-KD from in vitro translated lysates. Lower panel shows protein levels.