Figure 2.

EMSA of wt and mutant oligonucleotides containing the CUP- and/or Sp-(GT-box) binding sites with preadipocyte and adipocyte nuclear extracts. (A) ³²P-end-labeled EF oligonucleotide containing the CUP- and Sp-(GT-box) binding sites of the C/EBPα gene promoter (nucleotides −258 to −234) were incubated with 10 μg of nuclear extract from 3T3-L1 preadipocytes (day 0) or adipocytes (day 8). (B) wt ³²P-end-labeled EF oligonucleotide (EF), EF oligonucleotide mutated in the CUP-binding site (EF-MC), or EF oligonucleotide mutated in the GT-box were incubated with 10 μg of nuclear extract from 3T3-L1 preadipocytes. (C) Effect of wt and mutant competitor oligonucleotides and anti-AP2, -Sp1, and/or -Sp3 antibodies on EMSA with preadipocyte nuclear extract. Pi indicates preimmune serum. EMSA was performed with ³²P-end-labeled EF probe. Arrows (←←) indicate the position of supershifted DNA-protein complexes.