Activation of the canonical Wnt/β-catenin pathway is augmented by E6 proteins that can degrade NHERF1.

The listed E6 proteins were co-expressed with FLAG_E6AP_WT, the TOPFLASH or FOPFLASH luciferase reporter, and a renilla luciferase internal transfection control plasmid in C33A cells. Transfected cells were treated with Wnt3A conditioned media for 8.5 hours, lysed in 1X passive lysis buffer (Promega), and measured for luciferase and renilla luminescence. Fold activation was determined by normalizing the TOPFLASH luminescence by the FOPFLASH luminescence. Each E6 protein that could degrade NHERF1 (16E6_WT, 16E6_ΔPBM, 11E6_WT, and 18E6_WT) augmented the canonical Wnt pathway. 16E6_F69A/K72A, which cannot degrade NHERF1, failed to increase Wnt pathway activation over vector levels. Statistical significance was determined from three independent experiments by Student’s t-test (***<0.001, n.s. = no significance).