BCL11B binds and activates the IL-2 promoter through the US1 site. (A) EMSA using purified recombinant GST-BCL11B (lane 3) or GST (lane 2) and US1 (ii) or NFAT-AP1-Oct DNA probes (i). Lane 1 is free probe. (B) EMSA using nuclear extracts from Jurkat cells and the US1 probe. In the top panel the US1 probe was incubated with 5 μg nuclear extracts from Jurkat cells treated with PMA and ionomycin (lanes 1-3) and anti-BCL11B (lane 3) or nonspecific antibodies (lane 2). BCL11B-US1 complexes are indicated by the black arrow, and the antibody-supershifted BCL11B-US1 complex is indicated by the white arrow. In the bottom panel the US1 probe was incubated with extracts from cells treated with PMA/ionomycin or forskolin plus PMA/ionomycin. Treatments were conducted as described in Figure 6. (C) EMSA using purified GST-BCL11B (lanes 3) or GST (lanes 2) and proximal (ii) or distal (i) US1 DNA fragments. Lane 1 is free probe. (D) Mutational analysis of the BCL11B binding site. Top panel shows the sequence of the BCL11B binding site. Underlined are the bases mutated in each mutant (M1-M8). Middle panel shows EMSAs with purified recombinant GST-BCL11B, ³²P-labeled BCL11B binding site probe plus excess of unlabeled WT or mutant DNA competitors (M1-M8). The bottom panel represents the quantification of the relative intensity of the corresponding bands. Maximal binding of the probe was determined in the sample lacking competitor DNA. For simplicity EMSAs in panels A-D present only protein-DNA complexes. (E) Reporter assays from MSCV-BCL11B Jurkat cells transfected with the following IL-2 promoter-luciferase constructs: wild-type -210 (lane 1), wild-type -243 (lane 2), mutant A -243 (lane 3), and mutant AB -243 (lane 4) (see top panel). In each case cells were treated with PMA/ionomycin for 8 hours before harvesting. Three experiments were conducted, and the quantifications represent means ± SD.