FIG. 5.
Functional cooperation between Purα and YB-1 in transcriptional activation of the JCVCY minimal promoter in U-87MG cells. (A) pBLCAT3-CYE reporter plasmid containing the JCVCY minimal promoter (7.5 μg), as shown (top), was introduced into U-87MG cells alone or together with YB-1 and Purα expression plasmids. In cotransfections, as the plasmid concentration for one transactivator was kept constant at 10 μg for each lane (lanes 2 to 4 for YB-1 and lanes 7 to 9 for Purα), the plasmid concentration for the other transactivator was varied (5, 10, and 10 μg of plasmid DNA of Purα for lanes 3 to 5 respectively, and 5, 10, and 10 μg of plasmid DNA of YB-1 for lanes 8 to 10, respectively). One microgram of RSV-β-gal plasmid was added to each transfection mixture. The DNA concentrations for each lane were normalized with the addition of empty expression vector DNA. CAT activity was measured and normalized for β-gal activity. The transfection experiments were repeated at least three times. The data obtained from CAT assays were quantitated and are presented as fold activation relative to the basal expression of the minimal promoter (bottom). Error bars indicate standard deviations. Results of a representative CAT assay are shown in the middle. (B) Experiments similar to those detailed for panel A were performed with the JCV late promoter, pBLCAT3-CYL.