A beta-lactamase reporter assay for monitoring the activation of the smoothened pathway.
The seven-transmembrane protein Smoothened (Smo) mediates the cellular response to the Hedgehog protein signal and is involved in cell growth and differentiation during embryonic development. Stimulation of the Smo pathway is directly implicated in tissue maintenance and repair, but overactivation of Smo could lead to tumorigenesis. We developed a robust and sensitive functional cell-based assay that measures the activity of endogenous Smo using a beta-lactamase transcriptional readout. This is the first Smo reporter assay that utilizes beta-lactamase reporter technology. This assay type has distinct advantages over other reporter technologies and can be used in a high-throughput mode to search for therapeutically relevant downstream Smo target effectors.
Osak
Bella
B
Mechanistic and Target Biology, Pfizer, Ann Arbor, MI, USA.
Fitzgerald
Laura R
LR
D000818:Animals; D002452:Cell Count; D002460:Cell Line; D003001:Cloning, Molecular; D003627:Data Interpretation, Statistical; D004121:Dimethyl Sulfoxide / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical; D004789:Enzyme Activation; D005434:Flow Cytometry; D017930:Genes, Reporter / Q000235:genetics; D007202:Indicators and Reagents; D051379:Mice; D010957:Plasmids / Q000235:genetics; D043562:Receptors, G-Protein-Coupled / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D000072086:Smoothened Receptor; D014162:Transfection; D001618:beta-Lactamases / Q000235:genetics*
D007202:Indicators and Reagents; D043562:Receptors, G-Protein-Coupled; C491022:Smo protein, mouse; D000072086:Smoothened Receptor; D001618:beta-Lactamases; D004121:Dimethyl Sulfoxide