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.