Fig. 3. Identification of the first regulatory element (PRE1) required for v-Src induction of TGF-ß RII promoter. A, one copy of the PRE1 sequence from -219 to -180 was linked to adenovirus E4 minimal promoter (-38/+36)-luciferase reporter construct. WT, wild-type sequence. M5 and M7 possess the same sequences except for the 4-nucleotide substitution. M7 mutation contains the base substitution of AP1/ATF2-like sequence. These chimeric constructs were cotransfected with v-Src expression vector into HepG2 cell, which were harvested after 48 h and assayed for luciferase activity. Transfection efficiencies were normalized by ß-gal activity as described in "Materials and Methods." Results are averages of at least triplicate experiments of three independent transfections. B, electrophoretic mobility shift assay was performed with labeled PRE1 oligonucleotide and nuclear extracts of 32D myeloid cell lines. Competitions were performed with a 50-fold excess of the indicated unlabeled oligonucleotides. Lanes 1 and 6, free probe. Lanes 3–5 and Lanes 8–10, competition with unlabeled wild-type and mutant sequences in both cell lines. In Lanes 2 and 7, competitors were not added into the binding reaction. Arrows, specific bands increased by v-Src protein.