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I am reading an article ( wherein a TOPflash/FOPflash assay is used to detect beta-catenin protein levels in a COS-7 cell line. I can't find a good explanation in the article, or in any other article, as to how this assay works. I understand the cells are transfected with TOPflash/FOPflash reporter plasmids, but that's about it. Any insight or redirection would be much appreciated.

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From what I can understand, TOPflash is a luciferase reporter plasmid that contains two sets of 3 copies of the wild-type TCF binding regions. If the canonical Wnt signaling is activated, beta-catenin will translocate to the nucleus to associate with TCF/LEF transcription factors to activate transcription of Wnt target genes. Since this is a luciferase plasmid, you should see an increase in relative luciferase activity when substrate is added. If Wnt signaling is inhibited, you will see loss of luciferase expression. The FOPflash is used as a negative control because the TCF binding regions upstream of the luciferase gene is mutated so even if beta-catenin translocates to the nucleus to activate TCF-mediated transcription, you should not see any luciferase activity.

Hope this helps.

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Would you please provide a reference or two that supports your otherwise good answer. Good references helps to improve the overall quality of Biology.SE. :) – Mike Taylor Sep 18 '14 at 21:00

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