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Measurement of two caspase activities simultaneously in living cells by a novel dual FRET fluorescent indicator probe by Xiaoli Wu; James Simone; Derek Hewgill; Richard Siegel; Peter E. Lipsky; Liusheng He is a Biochemistry, Genetics and Molecular Biology article available to read on EtoBox.
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## Abstract ## Background: A number of fluorescent caspase substrates and FRET‐based indicators have been developed to study the in vivo activation of caspases, a conserved family of proteases critical in inflammatory, and apoptosis signaling pathways. To date, all substrates have measured only one caspase activity. Here, we describe a FRET‐based probe for simultaneously measuring two distinct caspase activities in living cells. ## Methods: This probe consists of a CFP‐YFP‐mRFP fusion protein containing a caspase‐3‐cleavage motif, DEVD, between CFP and YFP, and a caspase‐6‐cleavage site, VEID, between YFP and mRFP. DEVDase and VEIDase activities could be assessed simultaneously by monitoring diminished FRET mediated by cleavage of either or both of these protease cleavage sites using flow cytometry. ## Results: DEVDase and VEIDase activities were completely inhibited by the pan‐caspase inhibitor z‐VAD‐fmk and enhanced by DNA‐damaging drugs or by anti‐Fas stimulation. DEVD and VEID cleavage specificities were validated by using caspase‐3‐deficient MCF7‐Fas cells and a caspase‐6‐specific inhibitor. Kinetic analysis with the FRET probe revealed that caspase‐3 activation consistently p
Who reads Measurement of two caspase activities simultaneously in living cells by a novel dual FRET fluorescent indicator probe?
It is typically read by researchers, students, and practitioners in Biochemistry, Genetics and Molecular Biology.
- Author
- Xiaoli Wu; James Simone; Derek Hewgill; Richard Siegel; Peter E. Lipsky; Liusheng He
- Publisher
- John Wiley and Sons; Wiley (John Wiley & Sons); John Wiley & Sons Inc.; John Wiley & Sons Ltd.; Wiley (ISSN 0196-4763)
- Published
- 2006
- Language
- EN
- Field
- Biochemistry, Genetics and Molecular Biology (Life Sciences)