Human PDGF-D promoter analysis. (A) Putative Ets-binding sites (ggaa, ggat, ttcc, atcc) are indicated in bold letters. The 4 Ets-binding sites used in mutational analysis (Figure 3A, Ets-D1, D2, D3, D4) are shown by bold and underlined letters. One nonconsensus (n.c.) Sp1-binding site crossing with Ets-D3 is shown with frame and indicated as Sp1nc. The translational start site is italicized and bolded (CCAGCGC). The primers used for primer extension are shown by bold capital letters and labeled as primer A and B. Capital letters represent 5′-untranslated regions (UTRs). (B) Electrophoretic analysis of ³²P-labeled primer extension products. fX174 HinfI DNA markers (lane M) and the products from primer extension reactions using control and PDGF-D primers were separated by electrophoresis on an 8% denaturing polyacrylamide gel. Length (in base pairs) for each DNA HinfI fragment in the marker lane is indicated next to the corresponding band on the autoradiograph. In lanes 1 and 2 the source of the RNA is from early passage SMCs (passage 5) and lanes 3 and 4 from later passage (passage 8) SMCs. In lanes 1 and 3, primer A was used and in lanes 2 and 4 primer B was used. In lanes containing kanamycin-positive control RNA (lane 5 with 2 ng and lane 6 with 5 ng), the top band (87 bp) represents the major cDNA product using the control RNA. The bottom bands (25 bp) in lanes 2 through 6 represent the ³²P-labeled primers. The data in panel B are representative of at least 2 independent experiments. The arrow indicates a single 40-bp extension product using primer B.