Fig. 6. Kermit 2/XGIPC is required to maintain IGF1 induced phosphorylation of AKT in oocytes and animal cap explants. (A) Kermit 2 is required for maintaining IGF1-induced AKT phosphorylation in oocytes. Oocytes were injected with 40 ng of control morpholino (CM) or kermit 2 morpholino (K2M), cultured for 48 hours and then treated with recombinant human IGF1 protein (18 ng/ml) for 30 minutes or overnight. Kermit 2 is required for IGF1-induced AKT phosphorylation/activation after overnight treatment, but not for short-term phosphorylation of AKT or MAPK. Total AKT or total MAPK was used as the loading control. (B) Effects of kermit 2 morpholino on the endogenous activation of AKT and MAPK. Control morpholino or kermit 2 morpholino was injected into four animal blastomeres of four-cell/eight-cell embryos. Animal caps were explanted at stage 9 and harvested after 2 hours or overnight. Kermit 2 morpholino reduces endogenous AKT phosphorylation after overnight incubation but not after 2 hours incubation and does not affect MAPK phosphorylation at either time point. (C) Kermit 2 is required for IGF1-induced AKT phosphorylation in stage 20 animal cap explants. 500 pg of XIGF1 mRNA and 400 pg of GFP mRNA were injected into one-cell embryos and 40 ng of kermit 2 morpholino was injected into four animal blastomeres of four-cell/eight-cell embryos. Animal caps were dissected at stage 9 and harvested at stage 20. GFP was used as the injection control.