Figure 6.

Gel shift assays performed with nuclear extracts from cells transduced with PTEN or Akt expressing adenovirus. (A–C) Oligonucleotides corresponding to -88 to -66 base pairs of the human VEGF promoter were labeled with [γ-³²P]ATP. Gel shift assays were performed using nuclear extract from cells transduced with adenovirus expressing dead PTEN, wild-type PTEN, dominant negative Akt, myrAkt, or GFP as indicated in the headings above the figures. The DNA-binding reaction was performed with 100-fold molar excess of unlabeled competitor, Sp1, AP-2 consensus oligonucleotide as indicated. Cell line (SF188 or U87 MG) is indicated at top of gel. (A–C) Arrows point to shifted band. (D) Supershift gel shifts were performed using nuclear extract (5 μg) and 0.2 μg of AP-2 or HIF-1α (control) antibody. Arrow indicates position of supershifted complex. (E) Supershift gel shifts were performed using nuclear extract (5 μg) and 0.2 μg of Sp1, Sp3, or AP2 antibody. The position of supershifted complexes is indicated by arrow. (F) Lysates from U87MG cells were incubated with alkaline phosphatase for 15 min and then treated with phosphatase inhibitors in order to inactivate the enzyme. Then the radioactive probe was added, and gel shift assay was performed. Arrow points to shifted band.