Sp1 expression in Sp1-deficient SL2 cells showed that Sp1 transactivated <i>LEDGF/p75</i> promoter by direct binding to its sites.

<p>A, SL2 cells, a <i>Drosophila</i> cell line, were transfected with indicated amounts of pPac-Sp1 (lanes 2 and 3) or pPac-V (lane 1). The expression level of Sp1 protein was examined by Western blot. Relative band density is shown below (A, gray bar <i>vs</i> black bar). B, Increasing Sp1 expression selectively increased <i>LEDGF/p75</i> promoter activity in SL2 cells. SL2 cells were cotransfected with pPac-Sp1 or pPac-vector (pPac-V) and pCAT-<i>LEDGF/p75</i> wild-type (pCAT-LED) or its mutant (pCAT-LED-Mut) reporter plasmid or pCAT vector (pCAT-V). Cells were processed to assay CAT activity as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037012#s4" target="_blank">Materials and Methods</a>. Results were expressed relative to activity of the LEDGF/p75 reporter activity in the presence of pPac-V or pPac-Sp1 and are presented as histograms: pPac-Sp1 with WT promoter (gray bar), and pPac-Sp1 with mutant promoter activity (open bar). The results are mean ± S.D. of three independent experiments (** <i>p<0.001</i>). C, Sp1 directly and exclusively bound to its sites in <i>LEDGF/p75</i> promoter. Nuclear extract was isolated and processed and then incubated with radio-labeled DNA probe containing Sp1 site (Probe 1 or standard control probe, ^•Sp1). Nuclear extract from pPac-Sp1 overexpressed cells bound strongly to probe containing wild-type Sp1 consensus sequence (lane 2), but nuclear extract from pPac-vector transfected cells showed no binding with either wild-type or mutant probe (lanes 1 and 3). <i>Right</i> panel, Nuclear extracts of pPac-Sp1 transfected cells incubated with standard control probe containing Sp1 site (sc-2502, lane 4) or its mutant (sc-2503, lane 5) and nuclear extract from pPac vector transfected cells incubated with standard probe (lane 6).