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The cytomegalovirus (CMV) promoter-based expression vectors CMV-6, HA-Akt, and HA-Akt(K179M) have been described (6). HA-PH contains the coding region of HA-AH1 (24), including amino acids 1 through 147 of Akt, in CMV-6. The CMV-β-Gal expression vector was pON260 (36). All forms of Akt were tagged with a hemagglutinin (HA) epitope. Transfection of HA-Akt, but not HA(K179M), into COS cells (by DEAE-dextran) resulted in increased Akt activity relative to that in cells transfected with vector (CMV-6) (11). Cerebellar neurons were transfected in 24-well plates (Costar) by the calcium phosphate method on 4 or 5 DIV (or 4 or 6 DIV, in (29)] largely as described (36); cells were transfected in Dulbecco's modified Eagle's medium (DMEM) and then returned to complete medium. For some of the experiments of Fig. 5B, the glutamate receptor inhibitors kynurenate and D(-)-2-amino-5-phosphonovaleric acid (D-APV) were included in the transfection as described (36); however, these proved to be unnecessary and were subsequently omitted. Cells were stained as described (2); Cy-2 coupled antibody was from Biological Detection Systems. Scoring of transfected cells was done blinded with respect to the identity of the transfected plasmids. Transfectants were identified by immunostaining for the cotransfected β-Gal marker. This allowed comparison between empty vector and Akt transfectants. In addition, because β-Gal staining was relatively stable during apoptosis, staining for β-Gal avoids scoring bias toward healthy cells. For all neuronal transfections, a 2:1 DNA ratio of Akt expression vector to β-Gal expression vector was used, and thus all healthy cells expressing β-Gal also expressed transfected Akt (measured by anti-HA immunostaining); all Akt constructs showed similar intensity of staining.
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We thank J. Blenis, M. Weber, S. Pons, and M. White for antibodies; M. Kozlowski and J. Avruch for 40S ribosomal subunits; Amgen Inc. for BDNF; K. Auger and T. Roberts for assistance with the PI3-K activity assay; S. Vasquez for assistance with cerebellar cultures; L. Lipfert for assistance generating antibodies to Akt; and members of the Greenberg lab for critical reading of the manuscript and for technical assistance. T.F.F. acknowledges L. Cantley for continuing support. Supported by American Cancer Society grant PF4059 (H.D.), NIH grants DK39519 (M.J.B.), R01 CA43855 (M.E.G.), and R01 CA18689 (G.M.C.), Mental Retardation Research Center grant NIH P30-HD18655, a K. M. Hunter fellowship in Cancer Research from the National Cancer Institute of Canada (NCIC) (T.F.F.), NCIC grant 7168 (D.R.K.), and an H. E. Johns and Canadian Cancer Society Research Scientist Award from NCIC (D.R.K.). Animal care was in accordance with institutional guidelines.
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