Figure 7.

Nuclear CSD proteins bind to a supercoiling-induced single strand structure within the VEGF HR region. (A) Balb/c 3T3 fibroblast nuclear extract was preincubated with supercoiled plasmid DNA containing the VEGF HR region (pTKV5Luc), with the supercoiled backbone vector (pTK81Luc), with single strand self competitor (V5–) or left untreated (none). Extracts were then bound to labeled VEGF HR region single strand oligonucleotide (V5–) in the presence (+) or absence (–) of MgCl₂ in a gel retardation assay. MgCl₂ stabilizes supercoiling-induced single strand structures (27,39). Nuclear complexes (NF-V1, NF-V2 and NF-V3) are indicated. (B) Nuclear extracts were preincubated with restriction enzyme-digested (+) (relieves supercoiling and supercoiling-induced single strand structures) or undigested (–) (supercoiled) pTKV5Luc and pTK81Luc plasmids and bound to the labeled VEGF oligonucleotide (V5–) in a gel retardation assay.