Journal: Journal of Experimental & Clinical Cancer Research : CR
Article Title: Y-box binding protein 1 enhances DNA topoisomerase 1 activity and sensitivity to camptothecin via direct interaction
Figure Lengend Snippet: 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.
Article Snippet: DNA relaxation assays TOPO1 activity was measured by the relaxation of supercoiled pGEM-T easy plasmid (Promega, Madison, WI, USA).
Techniques: Activity Assay, Purification, Recombinant, SDS Page, Staining, Incubation, Binding Assay, Transfection, Western Blot