Research Paper Volume 1, Issue 9 pp 803—817

Figure 6. TTP binds E6-AP mRNA and targets it for rapid decay. (A) Schematic representation of E6-AP mRNA. The grey bar corresponds to E6-AP coding region and number-labeled black ovals represent putative 3' UTR AU-rich elements (AREs). m7G, 7-methyl-guanosine cap; AAAAn, polyadenylated tail. (B) E6-AP mRNA half-life was assayed in HeLa Tet-Off/TTP-Flag cells grown in the presence (triangles; labeled -TTP) or absence (circles; labeled +TTP) of Dox to induce TTP expression. After 48 hr, 5 μg/ml of actinomycin D was added to the cells and E6-AP mRNA decay was analyzed by qPCR using GAPDH mRNA as a normalization control. The data shown is the average of triplicate experiments. (C, D) Binding of TTP and E6-AP mRNA. Control and TTP-expressing (48 hr) HeLa Tet-Off/TTP-Flag cells were lysed and immunoprecipitation was performed on equal amounts of cytoplasmic lysates using control IgG or anti-Flag mAb. RNA purified from immuno-precipitates was subjected to RT-PCR (C) or qPCR (D) to detect E6-AP and GAPDH mRNA. The ethidium bromide-stained agarose gel depicting the 292bp E6-AP PCR product is shown in reverse image. The relative amounts of immuno-precipitated E6-AP mRNA is reported as the average 1/Ct value of triplicate experiments. (*) P < 0.01