FIG. 1.

Expression Akt isoforms in neonates, Akt1/Akt3 localization in E11.5 to E12.5 embryos, generation of Akt1/Akt3 compound, and double knockout mice. (A) Expression patterns of Akt isoforms in neonates. Four wild-type mice from two litters were sacrificed, and their tissues were pooled for lysate preparation. Forty micrograms of total protein was used for Western analysis with the indicated isoform-specific antibodies. (B) Akt1/Akt3 antibody specificity. Mouse Akt1, Akt2, and Akt3 cDNAs cloned in pCMV5 were expressed in HEK293 cells, and cell lysates were prepared for Western analysis. Immunoblot analysis was carried out with an affinity-purified Akt1/Akt3 antibody that recognizes Akt1 and Akt3. Actin levels were determined to control for equal loading. (C) Absence of Akt1 and Akt3 proteins in DKO mouse embryonic fibroblasts (MEFs). MEFs were prepared from wild-type, Akt1/Akt3 DKO, or Akt2/Akt3 DKO embryos, and cell lysates derived from these cells were used for Western analysis using the antibody as described in the legend to panel B with actin as control. (D) PCR genotyping for Akt1 and Akt3 loci. WT, wild type; DKO, double knockout. (E) Absence of Akt1 and Akt3 proteins in DKO mouse tissue (placenta). Total phospho-Akt is substantially decreased. (F to I) HE and immunohistochemical staining of E11.5 and E12.5 embryos with the Akt1/Akt3 antibody. Panels F and G are for the E11.5 embryo and H and I are for the E12.5 embryo; F and H show hematoxylin and eosin staining, and G and I show immunohistochemical staining. Akt1 or Akt3 (or both) is expressed in both the central and peripheral nervous systems of brain, neural tube, and heart. Arrowheads indicate various ganglia. Abbreviations: HB, hindbrain; MB, midbrain; FB, forebrain; BA, branchial arch; H, heart; NT, neural tube; CG, cranial ganglia; DRG, dorsal root ganglia; Li, liver; T, tongue.