Abstract
Background: The transcription factor NF-AT plays a key role in the activation of many early immune response genes and is regulated by subcellular localization. NF-AT translocates from the cytoplasm to the nucleus then returns in response to the intracellular calcium level. Methods: We have investigated NF-AT nucleocytoplasmic shuttling in real-time in living cells using NF-ATc1 tagged with the reversibly photoswitchable fluorescence protein, Dronpa. We monitored both nuclear import and export rate of Dronpa-tagged NF-AT in live cells upon stimulation with ionomycin plus calcium (I + Ca2+) or cyclosporin A (CsA). Results: The results show that NF-AT moved into the nucleus within 3-9 min after stimulation and moved back out into the cytoplasm within 15-50 min after CsA addition. In the absence of stimulation, NF-AT stayed in the cytoplasm as in the cells overexpressing GSK-3β, a calcineurin-opposing regulator. General Significance: This semi-quantitative imaging with constant fluorescence provides the basis to detect the real-time effect by several regulators on NF-AT family proteins.
Original language | English |
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Pages (from-to) | 1403-1407 |
Number of pages | 5 |
Journal | Biochimica et Biophysica Acta - General Subjects |
Volume | 1780 |
Issue number | 12 |
DOIs | |
Publication status | Published - 2008 Dec |
Keywords
- Calcineurin
- Dronpa
- GSK-3β
- NF-AT
- Nucleocytoplasmic shuttling
- Real-time imaging
ASJC Scopus subject areas
- Biophysics
- Biochemistry
- Molecular Biology