title: Capillary electrophoretic mobility shift assay for binding of DNA with NFAT3, a transcription factor from H9c2 cardiac myoblast cells.
abstract: 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 μM 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 7.7×10(9)  M(-1)  at 4°C.
authors.affiliation: Biomolecules Function Research Center, Korea Institute of Science and Technology, Seoul, Korea.
authors.forename: Soo Hyun
authors.initials: SH
authors.lastname: Park
authors.forename: Eunmi
authors.initials: E
authors.lastname: Ban
authors.forename: Eun Joo
authors.initials: EJ
authors.lastname: Song
authors.forename: Hyunjung
authors.initials: H
authors.lastname: Lee
authors.forename: Doo Soo
authors.initials: DS
authors.lastname: Chung
authors.forename: Young Sook
authors.initials: YS
authors.lastname: Yoo
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D004247:DNA / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D019075:Electrophoresis, Capillary / Q000379:methods*; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods*; D019332:Endothelin-1 / Q000737:chemistry / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000737:chemistry / Q000378:metabolism; D050778:NFATC Transcription Factors / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats
chemical_list: D019332:Endothelin-1; D050778:NFATC Transcription Factors; D004247:DNA