FIG. 8.

HPV-18 E6* reduces the half-life of ectopically expressed Dlg in 293 cells. (A) 293 cells were transfected with the construct expressing HA-tagged Dlg in the absence and presence of the construct expressing untagged HPV-18 E6*. After 24 h, cycloheximide was added to the dishes at 2-h intervals, and all the dishes were harvested for PAGE at 10 h. Residual Dlg levels were assessed by Western blot analysis. Short and long exposures of the Western blot are included for better comparison of the half-lives. Panel B shows the cumulative data from three experiments, analyzed using the densitometry analysis program in Adobe Photoshop, and then calculated as intensity relative to the time zero levels for Dlg expressed by itself or together with E6*. Standard deviations are indicated as vertical bars. (C) Determination of the half-life of HPV-18 E6*. Untagged HPV-18E6* was expressed in 293 cells, and after 24 h cells were treated with cycloheximide as for the experiments shown in panel A. Residual E6* levels were detected by Western blot analysis using anti-HPV-18 E6 monoclonal antibody 399. β-gal, β-galactosidase.