Figure 9.

Effect of myrAkt in DU145 prostate carcinoma cells. (A) Oligonucleotides corresponding to -88 to -66 base pairs of the human VEGF promoter were labeled with [γ-³²P]ATP. Gel shift assay was performed using nuclear extract from cells transduced with adenovirus expressing myAkt or GFP (control). The DNA-binding reaction was performed with 100-fold molar excess of unlabeled competitor, Sp1, AP-2 consensus oligonucleotide as indicated. (B) DU145 cells were infected with adenovirus expressing myrAkt or GFP. Thirty-six hours after infection, cells were in vivo–labeled with orthophosphate. In the top part of B labeled IP, in vivo–labeled proteins were immunoprecipitated with either Sp1 or Sp3 antibody as indicated. Immunoprecipitated complexes were separated on 10% SDS-PAGE gel and transferred to nitrocellulose membrane and autoradiographed. In the bottom part of B labeled IB (immunoblot), these same lysates were separated on 10% SDS-PAGE gel, transferred to nitrocellulose membrane, and probed with either Sp1 or Sp3 antibody to serve as a loading control.