ICSBP regulates the bcl-2 promoter. (A) Sequence of the mouse bcl-2 promoter (5′-UTR). Two forward (right arrow) and one reverse (left arrow) primer for amplification of promoter fragments are underlined. Boxes indicate IRF-target sequences: IRF/Ets composite element and GAS element. (B) Homology analysis between IRF recognition sequences in promoters of known IRF-target genes and the putative bcl-2 sites. GAS and Ets/IRF sites from various genes are shown, and conserved sequences are highlighted in underlined bold letters. The GAS- and Ets/IRF-specific consensus sequences are depicted. Boxes indicate Ets/IRF and GAS sites of the murine bcl-2 promoter. (C) Schematic representation of the 5′-UTR containing 2 IRF target sites as indicated. The maps of the short and long fragment promoter constructs or the empty control construct are shown. (D) Luciferase expression 48 hours after transfection of cells with short (left) or long (middle) bcl-2 promoter firefly luciferase construct, or (right) empty vector construct. Renilla luciferase was used as internal control and the firefly-renilla ratio was depicted. The mean (± SE) of 3 experiments is shown. (E) Detection of an ICSBP-dependent increase in sequence-specific protein-DNA complex formation by EMSA. Nuclear extracts from 32D/BA or 32D/BA-ICSBP cells give rise to 2 shifts as indicated (shifts I and II), with the ³²P-labeled EICE (EICE^*) from the bcl-2 5′-UTR used as a probe. Competition with cold DNA oligos (EICE oligo cold) (lanes 4-6) or mock oligo (lanes 7-9) was performed with excess cold oligo compared with ³²P-labeled probe (EICE^*) (20 ×,50 ×, and 100 ×). Mutated ³²P-labeled EICE (EICE^* mutated) was also used as a probe, identifying shift II as unspecific (lanes 7-10).