Luciferase assay for Wnt/b-catenin pathway - method misunderstanding (Mar/22/2009 )
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?
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.
Global Product Manager- Cellular Analysis
2800 Woods Hollow Rd.
Madison, WI 53711