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 35 and
Lanes 810, 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.