NANOG and KLF4 both bind to the NANOG promoter/enhancer and NANOG binds to the FLK1 promoter/enhancer in response to WNT3A. (A) HUVECs were starved of growth factors and serum, then left untreated or treated for 120 minutes with WNT3A (50 ng/mL). ChIP was performed with antibodies (Abs) specific for NANOG and KLF4, in addition to a nonspecific anti-rabbit IgG, as indicated (left). The left panel shows the PCR products (2.0 kb) from the human NANOG promoter/enhancer with the use of input chromatin prepared from HUVECs treated with or without WNT3A. (B) The primers used for amplification of the human NANOG promoter/enhancer region. (C) The relative positions of the primers used for amplification of the human FLK1 promoter region flanking the 8 putative NANOG binding sites. (D) Primers used for amplification of the human FLK1 promoter. (E) Interaction of NANOG with the FLK1 promoter. PCR product of the FLK1 promoter/enhancer (1.0 kb). (F) Biotinylated probes used for the EMSA. (G) Representative images of the EMSA experiment. Biotin-labeled oligonucleotides (probe 1 and probe 2) containing the sequences of the putative NANOG binding sites from the FLK1 promoter were incubated with nuclear extracts. The supershift was carried out by preincubation of the nuclear extracts with an anti-NANOG antibody. Note: Both monomeric and dimeric forms of NANOG interacted with the biotinylated probes. Arrowheads indicate the dimeric forms of NANOG in a native gel. Experiments were carried out ≥ 5 times.