Imaging Protein Kinase Cα Activation in Cells

Science  26 Mar 1999:
Vol. 283, Issue 5410, pp. 2085-2089
DOI: 10.1126/science.283.5410.2085

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Spatially resolved fluorescence resonance energy transfer (FRET) measured by fluorescence lifetime imaging microscopy (FLIM), provides a method for tracing the catalytic activity of fluorescently tagged proteins inside live cell cultures and enables determination of the functional state of proteins in fixed cells and tissues. Here, a dynamic marker of protein kinase Cα (PKCα) activation is identified and exploited. Activation of PKCα is detected through the binding of fluorescently tagged phosphorylation site–specific antibodies; the consequent FRET is measured through the donor fluorophore on PKCα by FLIM. This approach enabled the imaging of PKCα activation in live and fixed cultured cells and was also applied to pathological samples.

  • * These authors contributed equally to this work.

  • Present address: Synthelabo Recherche (LERS), 31 Avenue Paul, Valliant-Counturier, 92220 Bagneux, France.

  • To whom correspondence should be addressed. E-mail: (P.I.H.B.) bastiaen{at}, and (P.J.P.) parkerp{at}

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