Fig. 5
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Fig. 5
Appl1 Regulates Akt Activity, Akt Substrate Specificity, and Akt-Dependent Survival in Zebrafish Development
(A) Western blot analyses of Akt and MAPK level and activity in WT, Appl1 knockdown (MO: 8 ng MO1A), Appl1 overexpressing (mRNA: 75 pg appl mRNA), and rescued embryos (MO + mRNA: 8 ng MO1A + 75 pg appl mRNA).
(B) Forebrain apoptosis assay: Quantitative evaluation of apoptosis in Appl1 knockdown embryos rescued by expression of Akt (Akt rescue: 8 ng MO1A + 100 pg Akt2 mRNA). Embryos were assigned to one of the four indicated cell death categories. Evaluation of Appl1 knockdown and appl1 mRNA-rescued embryos (Figure 4G) are shown for comparison. Total amount of embryos scored: n Akt rescue = 396. Error bars indicate standard deviation (SD) amongst five experiments.
(C) Western blot analyses on the same fish extracts analyzed in panel (A) monitoring level and activity of Gsk-3β and Tsc2.
(D) Western blot analyses of MOCK and LY294002-treated fish. (Ser9)-Gsk-3β and (Thr1462) phosphorylation critically depends on Akt activity.
(A–D) Experiments have been carried out on 54 hr zebrafish embryos.
Reprinted from Cell, 133(3), Schenck, A., Goto-Silva, L., Collinet, C., Rhinn, M., Giner, A., Habermann, B., Brand, M., and Zerial, M., The endosomal protein Appl1 mediates Akt substrate specificity and cell survival in vertebrate development, 486-497, Copyright (2008) with permission from Elsevier. Full text @ Cell