期刊
BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS
卷 1810, 期 4, 页码 427-431出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.bbagen.2010.11.007
关键词
AhR; COX-2; c-Src; EGF; FRET; TCDD
资金
- National Institute of Environmental Health [R21ES15846]
- American Heart Association
- National Eye Institute [REY016754A]
- AHA [0665201Y]
Background: Activation of the protein tyrosine kinase c-Src (c-Src kinase) induced by the exposure to the environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been shown in various cell types. Most previous works used Western blot analysis to detect the phosphorylation on the Tyr416 residue, which activates c-Src kinase. Methods: Here we compared the results of c-Sic tyrosine phosphorylation via aryl hydrocarbon receptor (AhR)-dependent mechanisms from Western blot analysis with fluorescent resonance energy transfer (FRET) assay detecting c-Src activation after treatment with TCDD to activate AhR in two different human cell types. Results: Western blot analyses show time-dependent phosphorylation of c-Src by TCDD in HepG2 and MCF-10A cells. Data from FRET assay visualized and quantified the activation of c-Src kinase induced by TCDD in living cells of both cell types. The FRET efficiency decreased by 20%, 5 min after TCDD treatment and continued decreasing until the end of the experiment, 25 min after TCDD treatment. PP2, a c-Src specific inhibitor, suppressed both TCDD- and epidermal growth factor- (EGF) induced c-Src activation. In contrast, the AhR antagonist 3'-methoxy-4'nitroflavone (MNF) blocked only TCDD- but not EGF-induced activation of c-Src. Conclusions: The current study shows that the early activation of c-Src via EGF and AhR signaling pathways can be visualized in living cells using the FRET assay which is in line with Western blot analysis. General Significance: The FRET assay provides a useful tool to visualize and quantify c-Src kinase activation via AhR in living cells. (C) 2010 Elsevier B.V. All rights reserved.
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