Abstract
This protocol is for testing responses of a candidate cell line/cell lines to Hh ligands or Hh pathway agonists stimulation. This protocol can also be adapted to screen small molecule libraries or biologics that contain activities to either increase or decrease Hh pathway responses. Canonical Hh signaling activity transcriptionally induces Hh target genes that contain consensus Gli binding element. Hh-responsive cells transiently or stably expressing luciferase protein under the regulation of the Gli promoter element can be used to report stimulus-dependent Hh-pathway activity.
Materials and Reagents
Equipment
Procedure
Representative data
Figure 1. Firefly and renilla luciferase signals are measured at untreated and ShhN-treated conditions. Relative firefly/renilla signals are normalized as fold of induction to untreated conditions. Note: Relative Firefly Luciferase (FL)/Renilla Luciferase(RL) = Raw FL/Raw RL. Fold changes of FL/RL at Shh-stimulated condition is normalized to FL/RL at unstimulated condition.
Recipes
Acknowledgments
This protocol is adapted from published work (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
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.