Inhibition of repressor protein complex interaction with VWF DNA sequences in the presence of NFY binding oligonucleotide competitor.

Gel mobility experiments were carried out using a radioactively labeled oligonucleotide probe corresponding to DNA sequences +215 to +247 of the VWF gene. The probe (10 000 cpm) was incubated with nuclear extracts (5 μg) prepared from HeLa, BAE, and BSMC cells in the presence or absence of competitors and analyzed on a nondenaturing 5% polyacrylamide gel as described in “Materials and methods.” The competitors were double-stranded oligonucleotides corresponding to sequences +215 to +247 (ds-R, specific competitor), and sequences that contained E2A, C/EBP, and NFY consensus binding sites. Lane 1 contains the probe alone in the absence of nuclear extracts. The − represents the absence and the + represents the presence of 100× excess of each competitor. Samples containing nuclear extracts (5 μg) from each cell type are shown under the bold lines. Arrows show the position of the MC and the 3 faster migrating minor complexes C2, C3, and C4. The free probe is shown at the bottom of the gel. The figure shown is a representative of 3 independent experiments.