Fig. 5. The N-terminal extracellular region of Xfz3 is sufficient to induce dimerization. (A) Co-immunoprecipition of tagged N-terminal extracellular region of Xfz3. mRNA encoding extra3-myc and extra3-flag were injected alone or in combination in two-cell-stage embryos. Protein extracts from injected embryos were subjected to immunoprecipitation by using the anti-myc antibody (lanes 1 and 4) or the anti-flag antibody (lanes 2, 3 and 5). Immunoprecipitates were then immunoblotted with the anti-myc antibody. Extra3-myc is indicated with an arrow. (B) Analysis of Xbra expression in animal caps in response to the wild-type FGFR-1, a constitutive form of FGF-R1 receptor (torso-R1), Xfz3C-R1 or extra3-R1. Total RNA extracted from injected animal caps was assayed for Xbra expression at the early gastrula stage by RT-PCR. ODC is a loading control. RT–, control without reverse transcriptase. In each case, 50 pg of synthetic mRNA were injected. (C) Analysis of extra-R1 and Xfz3C-R1 phosphorylation. Embryos were injected with mRNA encoding the myc-tagged extra3-R1 or Xfz3C-R1 proteins. Protein extracts from the early gastrula stage were immunoprecipitated by the anti-myc antibody, separated by SDS-PAGE and blotted on nitrocellulose. Blots were analyzed with either anti-myc or anti-phosphotyrosine (4G10, UBI) antibodies.