Figure 5.

HMGB1 physically interacts with topoisomerase IIα. HMGB1, HMGB1-ΔC, domains A and B (expressed in E. coli and fused proteins with GST at their N-termini) were bound to glutathione-Sepharose beads (GST was used as a negative control). The beads were then incubated with isolated human topo IIα, followed by extensive washing of the beads, and boiling them in SDS-containing buffer. The released proteins were resolved by electrophoresis on an SDS–7.5% polyacrylamide gel, followed by western blotting and immunological detection using polyclonal antibody specific to topo IIα. Input, 10% of the total topo IIα used for the pull-down assay. Upper and lower PD experiments were performed in the absence or presence of 10 U of DNAse I in the incubation buffer, respectively.