| Chapter title |
Evaluating the Activity of Smoothened Toward G Proteins Using [ 35 S]Guanosine 5′-(3- O -thio)triphosphate ([ 35 S]GTPγS)
|
|---|---|
| Chapter number | 4 |
| Book title |
Hedgehog Signaling Protocols
|
| Published in |
Methods in molecular biology, January 2015
|
| DOI | 10.1007/978-1-4939-2772-2_4 |
| Pubmed ID | |
| Book ISBNs |
978-1-4939-2771-5, 978-1-4939-2772-2
|
| Authors |
David R. Manning, Feng Shen, Natalia A. Riobo, Manning, David R., Shen, Feng, Riobo, Natalia A. |
| Abstract |
The utilization of heterotrimeric G protein, and in particular those of the Gi, family, by Hedgehogs through Smoothened has become increasingly clear. We describe here a method for evaluating the activity of Smoothened toward G proteins in membranes derived from human embryonic kidney-293 (HEK293) cells. The assay relies on receptor-promoted exchange of GDP for [(35)S]GTPγS on the Gα subunit. The assay is best suited for analysis of the constitutive activity of Smoothened, inverse agonism superimposed on this activity, and neutral antagonism superimposed on inverse agonism. The assay would also be suitable for several other applications requiring a proximal, quantifiable readout of Smoothened activity. |
Mendeley readers
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| Unknown | 15 | 100% |
Demographic breakdown
| Readers by professional status | Count | As % |
|---|---|---|
| Professor > Associate Professor | 1 | 7% |
| Unknown | 14 | 93% |
| Readers by discipline | Count | As % |
|---|---|---|
| Agricultural and Biological Sciences | 1 | 7% |
| Unknown | 14 | 93% |