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Wnt/β-catenin luciferase reporter assay

CD Camille M. Drews
SC Samuel Case
SP Scott B. Vande Pol
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C33A cells plated at 70% confluency were transiently transfected with DNA of the TOPFLASH or control FOPFLASH (containing mutated TCF/β-catenin binding sites; 1 ug) plasmid, Renilla luciferase (0.005 ug) plasmid (used to evaluate transfection efficiency), FLAG_E6AP_WT (0.35 ug) plasmid, and the indicated E6 plasmids (0.3 ug). 18 hrs post-transfection, media was removed and Wnt3A conditioned media was added for 8.5 hours to stimulate the Wnt pathway. Luciferase levels were measured using the Dual-Luciferase Reporter Assay System (Promega) and a Cytation1 Plate Reader (software version 3.04.17). FOPFLASH luciferase readings were low, and were subtracted from the paired TOPFLASH readouts. 10% fetal bovine serum Wnt3A conditioned media was generated using L Wnt-3A murine fibroblasts (ATCC, CRL-2647) as previously described [64].

Copyright and License information:  ©2019 Drews et al
This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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