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Luciferase assay for Wnt/b-catenin pathway


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#1 LOgan

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Posted 22 March 2009 - 05:39 AM

Hi everyone,

I am using luciferase assay to investigate whether different factors can modulate the Wnt/b-catenin signaling pathway.
I am using topflash with the TCF/LEF binding sites upstream of the luciferase open reading frame, as a reporter for b-catenin activity.

As far as I am concerned, b-catenin needs the TCF/LEF complex of DNA-binding proteins to bind to DNA and activate target genes.
But, the topflash reporter is localized to the cytoplasm and the TCF/LEF DNA-binding proteins are localised to the nucleus. So, how can topflash report a cytoplasmic accumulation of b-catenin (which means an activation of the Wnt/b-catenin signaling pathway) if the rest of the complex (TCF/LEF) is in the nucleus?

Edited by LOgan, 22 March 2009 - 07:58 AM.


#2 KevinK

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Posted 27 March 2009 - 08:34 AM

LOgan,
I think one misconspetion helps celar this up. The luciferase reporter vector will be in the nucleus after transfection. The b-catenin translocates into the nucleus and transactivates TCF/LEF transcription factors (and creates increased luciferase signal in your assay).

Hope ths helps.

Kevin

Kevin Kopish
Global Product Manager- Cellular Analysis
Promega Corporation
2800 Woods Hollow Rd.
Madison, WI 53711
Promega Corporation
Madison, WI




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