Figure 8. C/EBP-binding sites (−470 to −459 bp and −404 to −390 bp) in the distal fragment are important for TGH promoter activity.

(A) EMSA was performed with nuclear extracts from 3T3-L1 adipocytes (day 5). A ³²P-labelled C/EBP consensus oligonucleotide was used as a probe. Unlabelled oligonucleotides (50×, 100×, 200× or 400×) derived from the TGH promoter (−470/−459 bp, 5′-TCTAAAGGTTGGTAAGTGTGAGTT-3′; −404/−390 bp, 5′-CAGGAAGGCTGTGAAATGTGTCCG-3′) or the C/EBP consensus [5′-TCTAAT(GT)(AGCT)(AGCT)G(AGCT)AA(GT)GAGTTC-3′] were added to the binding assays as competitors. The arrow shows the specific DNA–protein complex. The results are representative of two independent experiments. (B) Schematic representation of the different mutated reporter constructs (−542 Luc, −542 M1, −542 M2 and pGL3-Basic) used in transfection assays. Also shown are the results of trans-activation assays with different mutated TGH promoter–reporter constructs co-transfected with C/EBPα in 3T3-L1 pre-adipocytes. Values are means±S.E.M. of three independent transfections, normalized to co-transfected β-galactosidase. (C) Trans-activation assays of different mutated TGH promoter–reporter constructs transfected into 3T3-L1 adipocytes. Values are means±S.E.M. of three experiments.