Fig. 5. Slug binds to and is regulated by Ppa. (A) Slug was immunoprecipitated from lysates of embryos co-injected with tagged forms of Slug and Ppa using an -Myc antibody. Immunoprecipitates were resolved by SDS-PAGE and bound Ppa was detected by -Flag western (arrow). Interaction with Ppa is a conserved feature of Snail family proteins including those of Xenopus, mouse, and Drosophila Snail. (B) Slug was assayed for its ability to interact with the E3 ligase hcbl in co-IP experiments. Whereas Ppa efficiently binds Slug (arrow), hcbl does not. (C) Embryos were co-injected with Slug and control morpholino (con MO) or Ppa MO and allowed to develop normally (a) or were treated with CHX (b). The loss of Ppa mediated by the Ppa MO greatly stabilizes Slug protein. (D) Embryos injected with Slug alone or together with Ppa were treated with CHX at stage 8 and collected at the time points indicated. Slug protein is significantly destabilized by co-expression of Ppa. Actin is used as a control. (E) Embryos were injected in one of two cells at the two-cell stage with mRNA encoding Ppa and the lineage tracer, nß-gal (red, injected side to the left). Expression of Slug (a), FoxD3 (b) and Sox3 (c) were examined at stage 15 by in situ hybridization. Misexpression of Ppa results in failure of neural crest precursor formation and expansion of neural progenitors.