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Fig. 3. Sequences within the N terminus of Slug are responsible for its ubiquitin-mediated proteasomal degradation. (A) Schematic illustrating the mutant constructs used in these experiments. (B) Western blot demonstrating that the C-terminal zinc fingers of Slug are highly stable, but the stability of the N terminus is comparable to that of the intact protein. (C) Western blot analysis was used to monitor the relative stability of Slug with a series of Slug deletion mutants. When compared to the full-length protein, the stability of P38 was unchanged but the H64 mutant was greatly stabilized. (D) Western blot comparing the stability of Sox10 and a Slug N-Sox10 fusion protein. Addition of amino acids M1-L63 of Slug to Sox10 was sufficient to render it unstable (double bands represent a partial degradation product). (E) Poly-ubiquitinated forms of Slug were immunoprecipitated from lysates of embryos co-injected with tagged forms of Slug and ubiquitin. Slug is indicated by an arrow. In all blots an asterisk indicates IgG background band. (F) Embryos were co-injected with a mutant form of Ub (Ub^K48R) and either Slug, P38, or Slug-C and collected for western blot analysis. Whereas Slug and the unstable deletion mutant P38 both incorporate Ub^K48R (arrow), the stable C-terminal zinc fingers do not. (G) Western blot demonstrating the accumulation of higher molecular mass forms of wild-type Slug, but not the Slug-C or Slug P31-H64 deletion mutants, in response to treatment with the proteasomal inhibitor, MG132.

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