Figure 2.

CREB-1 is present in the DNA–protein complexes formed between forskolin-stimulated TF-1 nuclear extracts and identified CREs. EMS assays performed with forskolin-stimulated TF-1 nuclear extracts and radiolabeled probes corresponding to (A) LAI, (B) YU2, (C) 89.6, and (D) CXCR4 parental CREs revealed the presence of CREB-1 43-kDa protein in the DNA–protein complex (asterisk). Although the complex was unaffected by an isotype antibody, it was abrogated/supershifted with a CREB-1-specific antibody. Corresponding reactions with mutant TG CRE radiolabeled oligonucleotides exhibit the absence of the CREB-1-containing complex, suggesting that CREB-1 directly binds to parental CREs in vitro. Two other DNA–protein complexes (double and triple asterisks) are also present and are nonspecific in nature as they remain unaffected by addition of specific antibody.