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High-content tripartite split-GFP cell-based assays to screen for modulators of small GTPase activation
- Source :
- Journal of Cell Science
- Publication Year :
- 2017
- Publisher :
- The Company of Biologists, 2017.
-
Abstract
- The human Ras superfamily of small GTPases controls essential cellular processes such as gene expression and cell proliferation. As their deregulation is widely associated with human cancer, small GTPases and their regulatory proteins have become increasingly attractive for the development of novel therapeutics. Classical methods to monitor GTPase activation include pulldown assays that limit the analysis of GTP-bound form of proteins from cell lysates. Alternatively, live-cell FRET biosensors may be used to study GTPase activation dynamics in response to stimuli, but these sensors often require further optimization for high-throughput applications. Here, we describe a cell-based approach that is suitable to monitor the modulation of small GTPase activity in a high-content analysis. The assay relies on a genetically encoded tripartite split-GFP (triSFP) system that we integrated in an optimized cellular model to monitor modulation of RhoA and RhoB GTPases. Our results indicate the robust response of the reporter, allowing the interrogation of inhibition and stimulation of Rho activity, and highlight potential applications of this method to discover novel modulators and regulators of small GTPases and related protein-binding domains.<br />Summary: The development of a fluorescent reporter of GTPase activation based on tripartite split-GFP that enables the evaluation of GEF activity and the effect of modulators of GTPase activation in a high-content analysis.
- Subjects :
- 0301 basic medicine
RHOA
RHOB
Genetic Vectors
Green Fluorescent Proteins
GTPase
GTPase activation
03 medical and health sciences
0302 clinical medicine
Protein Interaction Mapping
Guanine Nucleotide Exchange Factors
Humans
Small GTPase
GEF
rhoB GTP-Binding Protein
biology
Cell-based assays
Cell Biology
High-Throughput Screening Assays
Tools and Resources
Cell biology
Protein–protein interaction
HEK293 Cells
030104 developmental biology
High-content analysis
030220 oncology & carcinogenesis
High-content screening
biology.protein
Split-GFP
GTP Phosphohydrolase Activators
Guanine nucleotide exchange factor
Cellular model
Ras superfamily
rhoA GTP-Binding Protein
Protein Binding
Subjects
Details
- ISSN :
- 14779137 and 00219533
- Database :
- OpenAIRE
- Journal :
- Journal of Cell Science
- Accession number :
- edsair.doi.dedup.....71d93ef6f7d0058664fc6fb451609f3b