Abstract
This protocol is for testing responses of a candidate cell line/cell lines to Wnt ligands or Wnt pathway agonists stimulation. This protocol can also be adapted to screen small molecule libraries or biologics that contain activities to either increase or decrease Wnt pathway responses. Canonical Wnt signaling activity transcriptionally induces Wnt target genes that contain concensus TCF/LEF binding element. Wnt pathway activity responsive cells transiently or stably expressing luciferase proteins under the TCF/LEF promoter element can be used to report stimulus-dependent Wnt-pathway activity. We acquired the TopFlash (TCL/LEF-Firefly luciferase) construct from Addgene.
Materials and Reagents
Equipment
Procedure
Representative data
Figure 1. Firefly and Renilla luciferase signals are measured at untreated and Wnt3a treated conditions. Relative firefly/renilla signals are normalized as fold of induction to untreated conditions. Relative Firefly Luciferase (FL)/Renilla Luciferase(RL) = Raw FL/Raw RL Note: Fold changes of FL/RL at Wnt3a-stimulated condition is normalized to FL/RL at unstimulated condition.
Recipes
Acknowledgments
This protocol is adapted from Kim et al. (2010). I thank the current and past members of the Beachy lab, Stanford University, who contributed to the development of this protocol. I acknowledge the Susan G. Komen for the Cure Postdoctoral Fellowship: KG111253.
References
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Hi,I got nice results from the experiment. I put my Wnt CM on transfected HEK 293T cells. Cells were transfected with TOP and FOP plasmids.