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Figure 2 | Molecular Medicine

Figure 2

From: Selective Inhibition of the Human tie-1 Promoter with Triplex-Forming Oligonucleotides Targeted to Ets Binding Sites

Figure 2

The activity of the two tandem Ets transcription factor core DNA binding sites, E-1 and E-2, in the human tie-1 promoter. (A) The effect of 5′ deletion on the activity of tie-1 luciferase reporter constructs (ptie-1luc) in endothelial cells. ptie-1luc constructs were cotransfected with a CMV promoter-driven Renilla luciferase control (pRL-CMV) plasmid into bovine aortic endothelial cells (BAECs). After a 72-h expression period, promoter activity was quantified by dual luciferase assay and normalized to pRL-CMV activity. Results are the means (± 1 SE, bars) of duplicate samples from six separate experiments (n = 12) expressed as a percentage of the activity of the 830-bp ptie-1luc construct. (B) Mutations (mut-1 and mut-2) were introduced into the putative ets core DNA binding motifs (EBS, 5′-GGAA/T-3′) in E-1 by overlap PCR as described in “Materials and Methods.” The top strand of the E-1 sequence from the human tie-1 promoter is shown. (C) The effect of Ets binding site mutations in E-1 on the activity of the human tie-1 promoter. Wild-type (tie-1), mutated (mut-1 and mut-2), and reverse tie-1 control (tie-1rev) luciferase constructs were cotransfected with pRL-CMV into BAECs. Promoter activity was determined by dual luciferase assay after a 72-h expression period and represented as a percentage of wild-type 880-bp ptie-1luc plasmid (tie-1) following normalization to pRL-CMV activity. Results are means (± 1 SE, bars) of duplicate samples from at least 3 separate experiments (n ≥ 6).

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