Study of TGF-β/Smad3 signal pathway using the technology of flurorescence resonance energy transfer

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 phosphoryl...

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Published inSheng wu yi xue gong cheng xue za zhi Vol. 31; no. 5; p. 1080
Main Authors Cao, Weiwei, Liu, Wei, Wang, Weishan, Chen, Zhao, He, Renhao, He, Jianwei
Format Journal Article
LanguageChinese
Published China 01.10.2014
Subjects
Fluorescence Resonance Energy Transfer
Genetic Vectors
HEK293 Cells
Humans
Microscopy, Fluorescence
Phosphorylation
Signal Transduction
Smad3 Protein - metabolism
Software
Transfection
Transforming Growth Factor beta1 - metabolism
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Summary: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 i
ISSN:1001-5515