Concurrent deletion of maternal Pten restored phosphatidylinositol 3-kinase–AKT signaling in two-cell EmPdk1+/−;Pten+/− embryos. (A) Levels of p-AKT (Thr 308), p-AKT (Ser 473), p-GSK3β (Ser 9), p-FOXO3a (Ser 253), p-TSC2 (Thr 1462), p-S6K1 (Thr 229), and p-S6K1 (Thr 389) were determined by using western blotting. Note that p-AKT (Ser 473) was recovered in EmPdk1+/−;Pten+/− embryos, restoring AKT activity as shown by the elevated levels of p-GSK3β (Ser 9), p-FOXO3a (Ser 253), and p-TSC2 (Thr 1462). Levels of p-S6K1 (Thr 229) and p-S6K1 (Thr 389) were not restored. (B) In vitro AKT kinase assay showing the restoration of AKT activity in two-cell EmPdk1+/−;Pten+/− embryos. FOXO3a, forkhead box O3; GSK3β, glycogen synthase kinase 3 beta; H2B, recombinant histone H2B; IP, immunoprecipitation; PI3K, phosphatidylinositol 3-kinase; S6K1, ribosomal protein S6 kinase, polypeptide 1; TSC2, tuberous sclerosis 2.