Bioluminescent imaging of MAPK function with intein-mediated reporter gene assay.
Methods Mol Biol
; 574: 185-92, 2009.
Article
en En
| MEDLINE
| ID: mdl-19685309
For nondestructive analysis of chemical processes in living mammalian cells, here we show a new reporter gene assay for detecting Ras-Raf-1 interactions based on protein splicing of transcription factors with DnaE inteins. The protein splicing induces connection of a DNA-binding protein (modified LexA; mLexA) with a transcription activation domain of a herpes simplex virus protein (VP16AD). Ras is connected with N-terminal DnaE and mLexA, while Raf-1 is connected with C-terminal DnaE and VP16AD. Upon stimulation with EGF, the interaction between Ras and Raf-1 triggers folding of the DnaEs, thereby inducing protein splicing to form mLexA-VP16AD fusion protein and transcription of a reporter gene, firefly luciferase. The extent of Ras-Raf-1 interaction is quantified by measuring the luciferase activity. By using the protein-splicing elements and the reporter gene, the Ras-Raf-1 interaction close to cell membranes can be evaluated.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Genes Reporteros
/
Proteínas Quinasas Activadas por Mitógenos
/
Inteínas
/
Luminiscencia
Límite:
Animals
Idioma:
En
Revista:
Methods Mol Biol
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2009
Tipo del documento:
Article
País de afiliación:
Japón
Pais de publicación:
Estados Unidos