Capillary electrophoretic mobility shift assay for binding of DNA with NFAT3, a transcription factor from H9c2 cardiac myoblast cells
- Capillary electrophoretic mobility shift assay for binding of DNA with NFAT3, a transcription factor from H9c2 cardiac myoblast cells
- 박수현; 반은미; 송은주; 이현정; 정두수; 유영숙
- capillary electrophoresis; Binding interaction; Cardiac hypertrophy; CEMSA; H9c2; NFAT
- Issue Date
- VOL 32, NO 16, 2174-2180
- Nuclear factor of activated T-cells (NFAT) is a transcription factor involved in the
development of cardiac and skeletal muscle and the nervous system. NFAT is activated by
calcium signal pathway and translocated into the nucleus. The quantification of binding
between NFAT and NFAT-specific DNA gives important information about cardiac
hypertrophy. We investigated the binding of NFAT3 in nuclear extracts from H9c2 cells
to its specific DNA by capillary electrophoretic mobility shift assay. The binding reaction
time required for stable formation of the DNA–NFAT3 complex was 3 h and the
separation of the complex and free DNA was achieved within 10 min by CE. The
formation of NFAT3–DNA complex was confirmed by the competitive reaction.
Comparison of the ratios of complex/free DNA peak area for 1 mM endothelin-1 (ET-1)-
treated cells and control cells showed the NFAT3 translocation into the nucleus
promoted by ET-1. The binding constant between NFAT3 and DNA was estimated to be
7.7 x 109M-1 at 41℃.
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