Figure 3

YB-1 promotes TOPO1 activity in DNA relaxation assays. A. Purification of recombinant proteins for DNA relaxation assays. Full-length YB-1 or YB-1 deletion mutants were expressed in bacterial cells, purified with 15 μl of glutathione-Sepharose 4B, and subjected to SDS-PAGE and Coomassie blue staining. B. Recombinant YB-1 promotes relaxation of supercoiled DNA. pGEM-T easy supercoiled DNA (0.25 μg) was incubated with TOPO1 (1 ng), GST, GST-YB-1 (40 or 400 ng) protein, or a combination of TOPO1 and GST-YB-1 for 30 minutes at 37°C (left panel). To test which part of YB-1 contained the TOPO1-binding domain, pGEM-T easy supercoiled DNA (0.25 μg) was incubated with TOPO1 (1 ng) alone, and TOPO1 (1 ng) with either GST, GST-YB-1, GST-YB-1 Δ1, GST-YB-1 Δ2, GST-YB-1 Δ3, or GST (400 ng) protein, for 30 minutes at 37°C (right panel). The DNA was resolved on agarose gels (without ethidium bromide), and stained thereafter with ethidium bromide. The supercoiled (sc) and relaxed (r) DNA bands are shown. C. Endogenous YB-1 knockdown with siRNA reduces TOPO1 DNA relaxation activity. PC-3 cells were transiently transfected with human YB-1 siRNA or control siRNA, and nuclear extracts (50 μg) were subjected to SDS–PAGE and western blotting. Transferred proteins were probed with anti-YB-1 and anti-TOPO1 antibodies, using anti-laminB1 antibodies as a loading control for nuclear protein (left panel). Forty-eight hours after the aforementioned transfection, various amounts of PC-3 nuclear extracts (NE) (1 μg to 4 μg of NE prepared by dilution in phosphate-buffered saline) were incubated with pGEM-T easy supercoiled DNA (0.25 μg) for 30 minutes at 37°C. The supercoiled and relaxed DNA bands are shown in the right panel.