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. 2012 Oct 18;287(49):41364–41373. doi: 10.1074/jbc.M112.377739

FIGURE 5.

FIGURE 5.

Binding of NF-κB p65 in the downstream of VEGFA gene. A, shown is a diagram of VEGFA downstream fragment #12 containing NF-κB p65 consensus site next to TBE-DS6. B, shown is ChIP analysis of TBE-DS within fragment #12. Anti-NF-κB p65 antibody was employed in addition to IgG control antiserum. Input DNA was also shown. HCT116 cells were grown in serum-free condition and activated by GW501516 (GW). Western blot (W.B) analysis was also performed to confirm the equal use of the input and immunoprecipitation of NF-κB p65 protein. Anti-β-actin antibody was used as a negative control. C, shown is ChIP-qPCR analysis with anti-NF-κB p65 antibody for NF-κB site in downstream fragment #12 (TBE-DS6). Three independent analyses were performed; the results represent the average ± S.D. (***, p < 0.005). NC denotes negative control without antibody. D, shown is RT-qPCR analysis of VEGFA mRNA after p65 overexpression in conjunction with GW501516 treatment. Expression of NF-κB p65 protein was confirmed by Western blot analysis. E, luciferase (Luc.) assay with the VEGFA promoter reporters with downstream fragment #12 (−2.3-kb VEGFA promoter + DS) with or without p65 overexpression. Three independent analyses were performed; the results represent the average ± S.D. (***, p < 0.005).