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Fig. 2 (A–C) Uninjected embryos (A), and embryos injected with WT march8 mRNA (B) or RING domain mutant (W109A) march8 mRNA (C), at shield to 60% epiboly stage. Overexpression of March8 results in loss of cell adhesion, abnormal cell migration, and cell death (B); individual embryos showing disaggregation of embryonic cells are shown in B2. Embryos injected with W109A mutant mRNA were normal (C). (D) Self ubiquitination of March8. HEK293T cells were transfected with the plasmids indicated and activated by addition of tebufenozide (see Materials and Methods). Lysates were immunoprecipitated (IP) with anti-March8 antibody and then immunoblotted (IB) with anti-HA antibody to detect ubiquitinated proteins (upper panel). Expression of March8 and tubulin as control are shown in bottom panels.