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Fig. 4. The RING domain is not absolutely required for the XPIASy activity. (A) XPIASy modifies a small ratio of XSmad2 with SUMO-1. The indicated mRNAs of Myc-tagged XSmad2 (0.5 ng), XPIASy (0.5 ng), flag-tagged SUMO-1 (0.5 ng) and/or P ${Delta}$ R (0.5 ng) were injected into blastomeres at the two-cell stage. Status of XSmad2 protein was analyzed at stage 10.5 by western blotting using Myc antibody. In addition, after immunoprecipitation with Myc antibody, the amount of SUMOylated XSmad2 was analyzed with Flag staining. (B) P ${Delta}$ R still inhibits mesoderm marker expression induced by XSmad2. The mRNAs of P ${Delta}$ R (0.5 ng) or/and XSmad2 (0.5 ng) were injected into both blastomeres at two-cell stage embryos. RT-PCR was performed using the animal caps as described in the Materials and methods. (C) P ${Delta}$ R still induces ventralized structures in a dose-dependent manner. The P ${Delta}$ R mRNA was injected into the DMZ at the four-cell stage at 1 ng (a), 3 ng (b) and 6 ng (c). The phenotypes were examined at stage 27. (d) Uninjected embryo.

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