Fig. 5. Identification of the second regulatory element (PRE2) required for v-Src induction of TGF-ß RII promoter. A, one copy of the PRE2 sequence from +2 to +36 was linked to adenovirus E4 minimal promoter (-38/+36)-luciferase reporter construct. WT, wild-type sequence. M4 possesses the same sequence except for the 5-nucleotide substitution of ETS binding site (+16 to +20). These chimeric constructs were cotransfected with v-Src expression vector into HepG2 cell, which were harvested after 48 h and assayed for luciferase. Results are averages of at least triplicate experiments of three independent transfections. ßgal expression vector (pRSV-ß-gal) was also cotransfected to normalize the transfection efficiencies. B, EMSA was performed with labeled PRE2 oligonucleotide and nuclear extracts of 32D myeloid cell lines. Competitions were performed with a 80-fold molar excess of the indicated unlabeled oligonucleotides. Lanes 1 and 6, free probe. Lanes 35 and Lanes 810, competition with unlabeled wild-type (S), nonspecific oligonucleotide (N), and M4 mutant sequence (M4) in two cell lines, respectively. Competitors were not added in Lanes 2 and 7. Unlabeled PRE1 element was used as nonspecific competitor. Arrows, specific bands in both cell lines. C, for antibody supershift assay, the oligonucleotide containing PRE2 element was incubated either with nuclear extracts alone of the 32D-src cell line (Lane 2), with normal rabbit IgG (Lane 3), with 0.5 µg anti-PU.1 antibody (Lane 4), or with 0.5 µg of anti-PU.1 antibody and 0.5 µg of competitor peptide against anti-PU.1 antibody, respectively. Supershifts with anti-PU.1 antibody are shown as indicated. PU.1 antibody, which is made against the DNA binding domain of the PU.1 protein, was purchased from Santa Cruz Biotechnology, Inc.