Figure 1.

Increase of SAF-1 DNA-binding activity in oncogenic ras-transformed MCF-10A cells. Nuclear extracts (10 μg of protein) prepared from untreated (lane 2), empty vector transformed (lane 3) or pCMV-rasV12 transformed (lanes 4–11) MCF-10A cells, as indicated, were incubated with ³²P-labeled VEGF DNA (−135 to +29) containing SAF-1-binding element. Lane 1 contains no nuclear extract. Resulting DNA-protein complexes were fractionated in a 6% nondenaturing polyacrylamide gel. In some assays, 50-fold molar excess of either nonspecific oligonucleotide (lane 6) or SAF-1-binding competitor oligonucleotide (lane 7) or normal IgG (lane 8) or antibody to SAF-1 (lanes 9 and 11) or antibody to Sp1 (lanes 10 and 11) were included during a preincubation reaction. Migration positions of SAF-1-specific complex are indicated. Super-shift (ss) of Sp1-specific DNA-protein complex is indicated. Complex A represents a DNA-protein complex present in normal MCF-10A cell nuclear extract is not affected by either SAF-1 competitor oligonucleotide, or the antibodies. SAF-1, serum amyloid A activating factor 1; VEGF, vascular endothelial growth factor.