Fig. 5

a Gel shift assay of cellular extracts incubated in vitro with oligonucleotides corresponding to a region of the VEGFR-2 gene promoter containing two putative HHEX binding sites. In some experiments, labeled oligonucleotide A1P1 containing two HHEX binding sites or labeled oligonucleotide A2P2 containing one HHEX binding site has been incubated with cellular extracts derived from PR9 cells grown either without additives (c) or for 24 h in the presence of Zn²⁺ or Zn²⁺ + ATRA. The specificity of the bands was tested adding a 100-, 200-, or 300-fold excess of unlabeled free oligonucleotide probe. b, c HHEX binding to the VEGF (b) and VEGFR-2 (c) promoters as shown by ChIP experiments. Nuclear extracts derived from PR9 cells grown for 24 h either in the absence (b) or in the presence of ZnSO₄ (Zn²⁺); cells were crosslinked in vivo; after cell lysis, chromatin fragments were immunoprecipitated with anti-HHEX antibody; and after DNA purification, DNA regions containing either VEGF or VEGFR-2 were amplified. In the figure, the VEGF or VEGFR-2 PCR signal without immunoprecipitation is indicated as −A (IgG control) and after immunoprecipitation as +A (HHEX antibody)