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Fig. 4 PH/G domain of Mtmr8 is required to cooperate with PI3K regulating embryo development and essential for the phosphatase activity of zebrafish Mtmr8 gene.
(A) Splice junction morpholino targeted against Mtmr8 exon-intron boundary resulted to loss of PH/G domain. RT-PCR of Mtmr8 transcript at 24hpf in WT and Mtmr8-MO2 (6 ng) morpholino-injected embryos, comparing cryptic spliced transcript in the morpholino injected embryos to the cDNA PCR products. (B) Embryo phenotypes observed under the microscope. LY represents the PI3K inhibitor, LY294002. Each picture represents typical results out of three separate experiments (30 embryos in each experiment). All scale bars are 100 μm. (C) Immunoblot of different extracts from Cont MO embryos and Mtmr8-MO2 morphants at 24 hpf (30 embryos/group). Relative p-Akt/Akt ratio was determined by band intensities that were analyzed by Scion software, with the ratio in experiment extracts normalized to the ratio determined in Cont MO injected embryo extracts. Results represent mean±SD of three separate experiments. *P<0.05.