EMSA using consensus Stat-binding site from FcγRI promoter.
(A) Nuclear extracts (5 μg) from peripheral blood T cells treated with medium alone (lane 1), PMA (lane 2), PMA/ionomycin (lane 3), PMA/ionomycin/CsA (lane 4), PMA/αCD28 (lane 5), and PMA/αCD28/CsA (lane 6) for 20 hours were used in EMSA. Nuclear extracts from preactivated T cells treated with medium alone (lane 7) and with IL-2 (100 U/mL; lane 8) were also used in the EMSA. 32P-labeled Stat-binding site from FcγRI promoter was used as a probe. Lanes 9 and 10 represent competition assays using an unlabeled oligonucleotide corresponding to the promoter sequence as a specific competitor (S.C.), and an oligonucleotide corresponding to the IFN-γ promoter sequence as a nonspecific competitor (N.C.). The arrowheads represent the specific bands, and the arrow indicates the nonspecific band. (B) Supershift assay using nuclear extracts from either preactivated T cells treated with medium alone (lane 1), and IL-2 (lanes 2-3), or T cells treated with medium alone (lane 4) and PMA/αCD28 (lanes 5-6). The arrow indicates the supershifted complex.
