FIG. 8.

CREB binds putative CRE sequences in the promoters of several adipocyte-specific genes. (A) The promoter regions of several adipocyte-specific genes were visually inspected for the presence of putative CRE sequences. Potential CREs present in these promoters are indicated by the box-enclosed regions which surround the nucleotides homologous to those in the consensus CRE sequences shown at the top of the figure. (B) Next, 20-bp double-stranded oligonucleotides, end labeled with [γ-³²P]ATP and polynucleotide kinase, were incubated with purified, recombinant CREB protein as described in Materials and Methods. The reactions were separated on nondenaturing, 6% polyacrylamide gels and exposed to Kodak X-ARomat film. The figure shows a representative autoradiogram of the free (bottom) and CREB-bound complexes in comparison to reactions performed with a nonspecific (NS) oligonucleotide lacking a CRE sequence. (C) Next, 5 μg of nuclear extract protein prepared from 3T3-L1 fibroblasts was incubated with the indicated, labeled oligonucleotides either in the absence (−) or presence (+) of CREB-specific antibody. The reactions were separated on polyacrylamide gels as described above and exposed to film. The figure shows a representative autoradiogram of unbound and protein-bound oligonucleotides. (D) A total of 5 μg of nuclear extract protein prepared from 3T3-L1 untreated (−) fibroblasts or cells treated with muristerone to induce KCREB expression (+) was incubated with the indicated, labeled oligonucleotides. The reactions were separated on polyacrylamide gels as described above and exposed to film. The figure shows a representative autoradiogram of unbound and protein-bound oligonucleotides.