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• _5 wasQuotedFrom 18483557 provenance.
• _5 value "Sequence analysis of the immunoprecipitated Tie2 and Foxf1 promoter regions confirmed the presence of several of the recently identified HOXA13 binding sites (Figure 10B) [71]. Next, using an electrophoretic mobility shift assay (EMSA), the HOXA13 DNA binding domain was confirmed to bind the promoter regions detected by the ChIP assay in a concentration-dependent manner (Figure 10C). Quantitation of HOXA13's affinity for the Tie2 and Foxf1 ChIP-positive regions using fluorescence polarization anisotropy revealed high affinity for the binding sites present in Tie2 (Kd = 27±1.4 nM and 22 nM±1.6 nM) and Foxf1 (Kd = 48±4 nM) compared to a control sequence lacking the HOXA13 binding site (Kd = 250±22 nM) (Figure 10D and 10E). Next, the capacity of HOXA13 to regulate gene expression through the 140 base-pair Tie2 and 121 base-pair Foxf1 ChIP-positive DNA fragments was examined (Figure 10F). The pGL3-Basic vector was selected for this analysis based on previous studies that confirm its capacity to assess promoter/enhancer activity in vitro, including previous characterizations of HOXA13's capacity to regulate transcription from minimal promoter elements [72], [74]–[78]. In the absence of the Tie2 or Foxf1 DNA elements, the empty pGL3-basic luciferase plasmid exhibited only a minor increase in luciferase expression when co-transfected with a Hoxa13 expression plasmid (Figure 10F). Similarly, the same luciferase vector containing either the Tie2 or the Foxf1 ChIP-positive regions also exhibited minimal luciferase expression in the absence of HOXA13 (Figure 10F). Co-transfection with a Hoxa13 expression vector stimulated luciferase expression from these minimal promoter elements resulting in low but significant increases in normalized luciferase expression: 3.7 fold for Tie2 and 3.2 fold for Foxf1" provenance.