Fig. 4. Identification of the inverted CCAAT box required for v-Src induction
of the TGF-ß RII promoter. A, site-directed mutations
were introduced in the inverted CCAAT sequence of pTßRIIP-132/+2,
which does not contain both PRE1 and PRE2 elements. WT,
wild-type sequence. R, right orientation of CCAAT box;
M, change of the inverted CCAAT sequence to AGGTT. These
constructs were cotransfected with v-Src expression vector into HepG2
cell, harvested after 48 h and assayed for luciferase activity.
Results are averages of at least triplicate experiments of three
independent transfections. ß-gal expression vector (pRSV-ßgal) was
also cotransfected to normalize the transfection efficiencies.
B, electrophoretic mobility shift assay was performed
with labeled oligonucleotide containing the inverted CCAAT box (-100
to -67; Table 1
) and nuclear extracts of 32D myeloid cell lines.
Competitions were performed with a 75-fold molar excess of the
indicated unlabeled oligonucleotides. Unlabeled oligonucleotide from
-100 to -67 was used as specific competitor (S).
Oligonucleotides of PRE2 elements (+2 to +36) was used as nonspecific
competitor (N). Lane 1, free probe.
Lanes 37, competition with unlabeled wild-type and
nonspecific oligonucleotides in both cell lines. Arrows,
specific bands in both cell lines. C, for antibody
supershift assay, the oligonucleotides containing the inverted CCAAT
box were incubated either with 10 µg of nuclear extracts of 32D
myeloid cell lines alone (Lanes 2 and 7),
with normal goat IgG (Lanes 3 and 8),
with 0.5 µg of anti-NF-YB (Lanes 4 and
9), or with 0.5 µg of anti-NF-YB and 0.5 µg of
competitor peptide against NF-YB antibody (Lanes 5 and
10), respectively. Supershifts with anti-NF-YB antibody
are shown as indicated.