Research Paper Volume 1, Issue 9 pp 803—817

Figure 5. TTP downregulates E6-AP mRNA and protein expression. (A) Northern blot (left panel) of TTP and E6-AP mRNA in HeLa Tet-Off/TTP-Flag cells 48 hr after TTP induction. RT-PCR assay (right panel) of HPV18-E6 and -E7 RNA levels in TTP-expressing cells. Actin and GAPDH were used as loading controls. (B) Western blot of E6-AP protein in HeLa Tet-Off/TTP-Flag cells (left panel) and adenovirus-infected HeLa cells (right panel) expressing TTP for 48 hr. Actin was used as a loading control. (C) TTP-dependent downregulation of E6-AP mRNA. HeLa Tet-Off/TTP-Flag cells were initially grown without Dox for 48 hr to induce TTP. At time zero, Dox was added to the culture medium and E6-AP mRNA levels were evaluated by qPCR over the indicated time course. E6-AP mRNA levels were normalized to control GAPDH mRNA. Cells grown in presence of Dox were used as control. All values shown are normalized to E6-AP expression of control-treated cells and are the averages of 3 experiments. (*) P < 0.01 (D) HPV 16-positive cells, SiHa (left panel) and CaSki (right panel) were infected with control AdGFP or AdGFP/TTP virus at an MOI of 100 or left untreated (NT). 48 hr after infection, cell lysates were examined for TTP, p53, and E6-AP expression by western blot. Actin was used as a loading control.