Abstract
Receptor activation upon ligand binding induces activation of multiple signaling pathways. To fully understand how these signaling pathways coordinate, it is essential to determine the dynamic nature of the spatiotemporal activation profile of signaling components at the level of single living cells. Here, we outline a detailed methodology for visualizing and quantitatively measuring the spatiotemporal activation of Ras and PKD1 by applying advanced fluorescence imaging techniques, including multichannel, simultaneous imaging and Förster resonance energy transfer (FRET).
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Acknowledgements
The authors would like to thank all members of the Chemotaxis Signal Section. This research was supported by the Intramural Research Program of the NIH, NIAID. We thank Dr. Derek C. Braun at the Gallaudet University, Washington, D.C. for his assistance in construction of PKD1 -CY and John F Hancock for sharing the plasmid of Ras -mRFP for this work. We also thank Howard Boudreau and Thomas Leto for providing MDA-MD-231 cells for this work. Q. Jane Wang was supported in part by National Institutes of Health grants R01CA129127 and R01CA142580.
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Xu, X. et al. (2016). Quantitative Monitoring Spatiotemporal Activation of Ras and PKD1 Using Confocal Fluorescent Microscopy. In: Jin, T., Hereld, D. (eds) Chemotaxis. Methods in Molecular Biology, vol 1407. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3480-5_22
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DOI: https://doi.org/10.1007/978-1-4939-3480-5_22
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