Measuring Smoothened (SMO)-Mediated Activation of the Gi Protein.

The oncoprotein Smoothened (SMO) can transduce the Hedgehog signal from the tumor suppressor Patched-1 (PTCH1) to glioma-associated-oncogene (Gli) transcription factors. Previous studies have shown that SMO is a G-protein-coupled receptor (GPCR) of the Frizzled-class (class-F) that can activate Gi family of heterotrimeric G proteins. Here, we describe [35S]-GTPγS assay using SMO cell membranes and purified Gi protein to measure the level of Gi protein activation following the activation of SMO by agonists.