FIG. 5.

Stability of YB-1 protein is increased in the presence of T-antigen. U-87MG cells were either transfected with CMV–T-antigen alone (lane 3) or cotransfected with pEBV-His-YB-1 and CMV–T-antigen expression plasmids (lanes 6 and 7). Lanes 4 and 5 received only pEBV-His-YB-1. Expression plasmid concentrations used in the transfections are indicated above the panels. The DNA concentrations for the transfection mixtures were normalized by addition of the respective empty expression vector DNA. A flag-tagged CDK9 expression plasmid was also included in the transfection mixture as an internal control for transfection efficiency. Thirty micrograms of whole-cell lysate was resolved by SDS–10% PAGE followed by immunoblotting. The different strips were cut out from the same blot and analyzed for T-antigen (T-Ag), His-tagged YB-1, and flag-tagged CDK9. Whole-cell lysate from untransfected cells (lane 1) was loaded as a negative control. The arrowheads indicate nonspecific bands. The arrows indicate the positions of the respective proteins. The positions of molecular mass markers (in kilodaltons) are shown to the left of the panels.