Fig. 4.

Characterization of the V2 exon regions required for achieving maximal V2 promoter activity. A and B, V2 promoter reporter constructs with progressive deletions of the 3′ V2 exon region were prepared and transfected into COS-1, HEK293, and Huh7 cells (A) and SGBS preadipocytes and mature adipocytes (B). Data are presented as mean ± se from n = 3–14 experiments. A significant decrease in promoter activity was observed from +162 to +103 for all five cell types. *, P < 0.05; **, P < 0.01; ***, P < 0.001. C, 3′-Deletion constructs covering the region from +162 to +103 were generated and transfected into HEK293 cells. Deletion of the region from +162 to +125 in the V2 exon caused the reporter activity to decrease significantly (***, P < 0.001). Data are expressed as mean ± se of n = 10 independent experiments. D, A double-stranded oligonucleotide probe containing the +123 to +144 sequence was end-labeled with [γ-³²P]ATP and incubated with nuclear extracts prepared from HEK293 cells for EMSA. For oligonucleotide competition experiments, a 100-fold (lane 3) or a 200-fold (lane 4) excess of unlabeled probe was added before the addition of the labeled probe. The three specific DNA-protein complexes are indicated by arrows.