Element D3 mediates basal and Ets-1–inducible PDGF-D promoter activity and binds both endogenous and recombinant Ets-1 and Sp1. (A) Mutational analysis of Ets-binding sites. SMCs were cotransfected with 1 μg pcDNA3-Ets-1 or pcDNA3 plasmid together with 10 μg p1168PDGF-Dluc or p1168PDGF-Dluc bearing mutated Ets-binding sites D1-4. The locations of the mutations in the PDGF-D promoter are shown in Figure 1A. (B) EMSA (left and top right panels) was performed by incubating 32P-OligoEts-D3–481/–457 bearing wild-type or mutant Ets element D3 and nuclear extracts of SMCs. A 10 × and 50 × molar excess of unlabeled Oligo Ets-D3–481/–457 wild-type or mutant type Oligo Ets-D3–481/–457 was used in competition analysis. Polyclonal antibodies to Sp1, Ets-1, or p53 (0.5 μg) were incubated for 15 minutes prior to the addition of probe. SNC denotes specific nucleoprotein complex. ChIP analysis (right panels) was performed using 2 separate primer sets with antibodies to Ets-1 or Sp1 or no antibody. Antibodies to Ets-1 and Sp1 pulled down the PDGF-D promoter, which was then amplified to produce amplicons of 360 and 390 bp, respectively. The omission of an antibody did not support amplification of any product. NE indicates nuclear extracts. (C) Ets element D3 binds recombinant Ets-1 and Sp1. EMSA was performed with 32P-OligoEts-D3–481/–45732P-OligoEts-D3–481/–457 containing mutant Ets-D3 or Ets–D3-2 and recombinant Ets-1 (100 ng) or Sp1 (100 ng). Free probe was run off the gel. The data are representative of at least 2 independent experiments. Error bars represent SEM performed in duplicate or triplicate. *P < .05 relative to control using Student t test.
