Figure 7. Sp1 expression in Sp1-deficient SL2 cells showed that Sp1 transactivated LEDGF/p75 promoter by direct binding to its sites.

A, SL2 cells, a Drosophila 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 vs black bar). B, Increasing Sp1 expression selectively increased LEDGF/p75 promoter activity in SL2 cells. SL2 cells were cotransfected with pPac-Sp1 or pPac-vector (pPac-V) and pCAT-LEDGF/p75 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 Materials and Methods. 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 (** p<0.001). C, Sp1 directly and exclusively bound to its sites in LEDGF/p75 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). Right 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).