Fig 1. Overexpression of EC1-3-myc and treatment with recombinant EC1-3 increases Akt phosphorylation.

(A, upper) Injections of 200 pg mRNA for UGP alone or together with 800 pg of EC1-3-myc mRNA were performed on single blastomeres of 2-cell X.laevis embryos. CNC expressing UGP were dissected at neurula stages, lysed and analyzed for phospho-Akt (pAkt), phospho-MAPK (pMAPK) and GAPDH via western blot. (A, lower) Non-injected CNC were dissected and treated for 5 minutes with 10 ng/mL of recombinant EC1-3 (bacEC) before being processed as above. (B) As a control, bacEC was depleted from treatment solution using a specific bacEC antibody conjugated to agarose beads and then applied to CNC. Lysates were then analyzed as above. Immunodepletion using unconjugated beads was used as a control. (C) Significant increases in phospho-Akt were observed in CNC overexpressing EC1-3-myc (N = 5, p = 0.013) and those treated with bacEC (N = 5, p = 0.021). There were not significant (N.S.) increases in the phosphorylation of MAPK in CNC overexpressing EC1-3-myc (N = 5, p = 0.09), nor those treated with bacEC (N = 5, p = 0.178). GAPDH was used as a loading control for western blots and normalizer for calculation of phospho-Akt and phospho-MAPK. Error bars are one standard deviation to the mean. One-tailed, Student’s t-tests were performed to determine statistical significance. * p<0.05. N, number of experiments.