Study of TGF-β/Smad3 signal pathway using the technology of flurorescence resonance energy transfer / 生物医学工程学杂志
Journal of Biomedical Engineering
;
(6): 1080-1084, 2014.
Artigo
em Chinês
| WPRIM
| ID: wpr-234454
ABSTRACT
The transforming growth factor-β1 (TGF-β1)/Smad3 signal pathway is related to mutiple physiological and pathological generation mechanism of human being. Up to date, however, the spacial and time information on the phosphorylated Smad3 is still unclear. In this study, the process of Smad3 phosphorylation was observed under the physiological state in the living cells. Firstly, the ECFP-Smad3-Citrine (Smad3 biosensor) fusion protein expression vector was constructed and identified. Then the Smad3 biosensor was transfected into 293T cells. The transfection efficiency and the expressions of fusion proteins were observed in 24 hours. Thirdly, Smad3 biosensor flurorescence resonance energy transfer (FRET) was observed with the inversion fluorescence microscope and measured by the MetaFlour FRET 4. 6 software. Smad3 biosensor transfection efficiency was nearly 40% and the fusion protein was seen under the fluorescence microscope. The FRET ratio of Smad3 biosensor in living 293T cells was decreased after 10 minutes incubation with the ligand of TGF-β1. The period of decreasing CFP and enhancing Citrine signals was about 300 seconds. With the technology of FRET, the TGF-β1/Smad3 signal pathway could be real time monitored dynamically under the physiological condition in living cells.
Texto completo:
DisponíveL
Índice:
WPRIM (Pacífico Ocidental)
Assunto principal:
Fosforilação
/
Software
/
Transfecção
/
Transdução de Sinais
/
Transferência Ressonante de Energia de Fluorescência
/
Proteína Smad3
/
Fator de Crescimento Transformador beta1
/
Células HEK293
/
Vetores Genéticos
/
Metabolismo
Tipo de estudo:
Estudo prognóstico
Limite:
Humanos
Idioma:
Chinês
Revista:
Journal of Biomedical Engineering
Ano de publicação:
2014
Tipo de documento:
Artigo
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