Nef inhibits Tcf –luciferase reporter activity.

<p>A. Promoter negative control: HEK293 cells were transfected with the TopFlash or the FopFlash reporters and with 50 ng Nef encoding plasmid (WT-Nef, the indicated mutants or empty vector) and incubated with Wnt-conditioned media (Wnt-CM) or control media that contain no Wnt. The TopFlash plasmid has TCF binding sites that are mutated in the FopFlash plasmid. The sites in FopFlash do not interact with TCF and therefore FopFlash serves as a negative control. Renilla luciferase was used for normalization purposes. Data shown here represent two experiments, each done in triplicate. WT-Nef significantly (P<0.00001, t-test) inhibits TCF reporter activity as compared to empty vector and also compared to the β-catenin motif mutants, D186A-Nef and F191A-Nef (P<0.00001, t-test) when co-transfected with TopFlash. B. The firefly/renilla values from panel A of this figure were used to calculate the TopFlash/FopFlash ratios of each condition. The resultant TopFlash/FopFlash ratios were then used to calculate the percent inhibition by WT-Nef and the indicated mutants on transcription in cells stimulated with Wnt.