Figure 4

NFAT molecules bind to the TR2 gene promoter in EL-4 T cells, and TR2 promoter activity is affected by dnNFAT and siRNA. (A) EMSAs were performed using nuclear extracts from EL-4 T cells without treatment with PMA/ionomycin (lane 1), and supershift assays were performed using anti-NFATc1, c2, c3, c4, and c5 (lane 2, 3, 4, 5, and 6). Arrows identify retarded complexes putatively formed by NFATs (C1, C2, C3). (B) Recruitment of NFAT components measured in nuclear extracts of EL-4 T cells activated with PMA/ionomycin for 15 min (lane 2-5), 5 h (lane 6-9), and 20 h (lane 10-13). The letters c1, c2, c3 refer to anti-NFAT c1, c2, and c3 antibodies, respectively. (C) TR2 promoter activity was measured using the luciferase reporter plasmid pTR2(-276/-3) co-transfected into EL-4 T cells with various concentrations of dnNFAT; as a control, dnNFAT was omitted. Cells were harvested 24 h after transfection, and luciferase activity was measured with the dual luciferase assay system (Promega). It was normalized to β-galactosidase activity and is expressed as percentages of the activity with Wt in the absence of dnNFAT. (D) RNA knockdown experiments were performed. EL-4 cells were transfected by electroporation with a siRNA duplex and the TR2 promoter-driven luciferase construct. The transfection efficiency of the siRNA was measured by Western blot analysis using mAb for NFATc2 (Top). TR2 promoter activity was measured using the luciferase reporter plasmid pTR2(-276/-3) cotransfected into EL-4 T cells with NFATc2-specific siRNA; as a control, nonspecific siRNA was used (Bottom). Luciferase activity was measured as in C.