Fig. 3. Cul1-C75 and Cullin-1 RNA injections increase the level of ß-catenin in the Xenopus embryo. (A) 1.6 or 2.5ng of Cul1-C75 (truncated) capped RNA were injected animally into each blastomere of two-cell stage X. laevis embryos. Proteins were extracted at stage 10 and analysed for ß-catenin levels. Injection led to an upregulation of ß-catenin levels. Blots were stripped and analysed against -tubulin as a loading control. (B) The same was carried out for full-length Cullin-1 RNA. Embryos were analysed at stage 8 and surprisingly an upregulation of ß-catenin levels was also observed. (C) ß-Catenin levels were also analysed at stage 19 (neurula stage), a time during which neural crest tissue becomes specified. As at stage 8 and 10, injections of 1.6 and 2.5 ng of either the Cul1-C75 and the full-length Cullin-1 lead to an increase in ß-catenin levels. This was also found for stage 24 (data not shown). To determine whether both constructs are translated correctly, western blots were stripped and subsequently analysed for Cullin-1 protein levels (with an antibody raised towards Cullin-1's N-terminus). The full-length Cullin-1 RNA (lane 4 and 5) results in higher levels of proteins of the correct size when compared with uninjected embryos (lane 1). A clear size difference between Cul1-C75 and Cullin-1 is also visible. (D) A wide range of Cullin-1 RNA doses were injected (10 pg-3 ng into each blastomere at the two-cell stage) and embryos were analysed at stage 10. A decrease in ß-catenin was never observed; indeed the increase of ß-catenin correlates with the increased dose of Cullin-1 injection.