Figure 4. Nuclear extract from LECs bound to Sp1 sites present in human LEDGF/p75 promoter.

A, Representative gel-shift mobility assays showing Sp1 binding to radiolabeled oligonucleotide probes containing consensus Sp1 sites as indicated. Nuclear extracts isolated from hLECs were incubated with ³²p-labeled probes containing Sp1 binding sites (WT-probes) or their corresponding mutants (Mut probes). Nuclear extracts bound to oligos containing Sp1 sites and yielded to complex, Sp1/DNA (Cm1) (A, lanes 1, 3 and 5). No complex occurred with mutant probes (A, lanes 2, 4, and 6). The oligonucleotide probes of both wild-type and mutated sequence used in assay are shown adjacent to image. B, Gel-shift assay showing the binding of Sp1 in nuclear extract of Sp1 overexpressed with hLECs to ³²p-labeled probes with its site. Nuclear extract isolated from cells transfected with plasmid encoding Sp1 or its corresponding vector was incubated with WT-probe1 or standard control probe (sc-2502; Santa Cruz Biotech). The DNA-protein complex was resolved on a 5% acrylamide gel. A discrete Sp1 expression-dependent DNA-protein complex was observed (B; lanes 1 vs 3) in comparison to vector transfected cells (lane 1), while the mutated probe failed to generate the complex (B, lanes 2 and 4). B, Right (lanes 5 and 6), depletion of endogenous Sp1 with its specific antibody. Nuclear extracts were incubated with either anti-Sp1 antibody (lane 6) or normal rabbit IgG (lane 5), and recovered nuclear extracts were incubated with the same probes (lanes 5 and 6). Lanes 7 and 8, standard control containing ^•Sp1 site (sc-2502, Santa Cruz Biotech) or its ^@mutant (sc-2503) processed for gel-shift assay using the same nuclear extracts. Extreme right, Depletion assay using anti-Sp3 antibody with nuclear extract showing no change in Sp1/DNA complex (lane 10) and the complex was indistinguishable from Lane 9. Images are representatives from three independent consistent observations.