Fig. 6.

The E7 Rb binding domain is necessary for the increased half-lives of MRN complex components in HPV31 positive cells. (A, B) Uninfected HFKs, HFKs maintaining wild type HPV31 genomes (HFK-31), HPV31 genomes containing a mutation in the E7 Rb binding site (HFK-31 ΔLHYCE), as well as (C, D) HFKs stably transduced with a retroviral vector expressing wild type E7 (pLXSN-31 E7), or E7 with a mutation in the Rb binding domain (pLXSN-31 E7 ΔLHCYE) were treated with 50 μg/ml cycloheximide over 12 h time course, and whole cell lysates were harvested at the indicated time points. Western blot analysis was performed using antibodies to MRE11, Rad50, and NBS1, as well as GAPDH, which served as a loading control. (A, B) Data shown are representative blots from three independent experiments from two different HFK donors, and for (C, D) data shown are representative blots from three independent experiments from one HFK donor. (B, D) Graphed are the average protein levels of MRE11, Rad50, and NBS1 over three independent experiments. Westerns were digitally imaged using the Bio-Rad Chemidoc MP system, and densitometry was performed with the Biorad ImageLab 5.0 software. Values are shown relative to each T0, which was set to100. Error bars represent +/− the standard error of the mean.