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. 2006 Aug;80(15):7339–7353. doi: 10.1128/JVI.00309-06

FIG. 6.

FIG. 6.

Transactivation activity of IE62d20 mediated by USF. (A) Immunoblot analysis using polyclonal anti-IE62 antibody to detect expression of IE62 and IE62d20 in MeWo cells. Sixteen micrograms of pCMV62 and pCMV62d20 plasmids was transfected into MeWo cells in 100-mm petri dishes. Forty-eight hours posttransfection, cell extracts of the transfected MeWo cells were isolated and resolved by SDS-PAGE. (B) Results of transient-transfection assays. One microgram of pRFL/WT and pRFL/Sp1sub reporter vector was cotransfected with or without 0.02 μg pCMV62 and pCMV62d20 into MeWo cells. Striped bars represent the luciferase activities derived from the reporter vector alone, which were normalized to 1. Solid bars represent the wild-type IE62-mediated activities, and the open bars represent the IE62d20-mediated activities, both of which are reported as induction (n-fold) of the luciferase activity over the basal level. Data represent the averages of triplicate transfections. The error bars indicate standard deviations. (C) Results of coimmunoprecipitation experiments using anti-IE62 monoclonal antibody and extracts derived from cells transfected with pcDNA, pCMV62, and pCMV62d20. Detection of the levels of wild-type and mutant IE62 and USF1 was performed by immunoblotting using rabbit polyclonal antibodies against the respective proteins. (D) Positions and sequences of the 5- and 10-bp insertions between the ORF28 TATA element and the USF site within the ORF28/29 regulatory element. The TATA element and USF site are shown in italics. The insertions are shown in lowercase and underlined. The designations of the resulting dual luciferase reporter vectors are listed at the left. (E) Results of transfection assays showing the effects of the insertions on transactivation by IE62 (solid bars) and IE62d20 (open bars) in the context of the Renilla luciferase (ORF28) reporter. (F) Results of transfection assays showing the effects of the insertions on transactivation by IE62 (solid bars) and IE62d20 (open bars) in the context of the firefly luciferase (ORF29) reporter. Data in panels E and F represent the averages of triplicate transfections. The error bars indicate standard deviations. Statistical significance of the differences observed with IE62 and IE62d20 was determined by one-way analysis of variance followed by Tukey's post hoc test.