Figure 2

Identification of the TOPO1 binding domain in YB-1. A. Schematic illustration of the GST-YB-1 deletion mutants (Ise et al., 1999 [26]). CSD indicates the cold shock domain. Amino acid residues are numbered. B. Interaction of GST-YB-1 deletion mutants with ThioHis-TOPO1. Approximately 500 μg of each GST fusion protein was immobilized on 15 μl of glutathione-Sepharose 4B, and the resin was incubated with 500 μg of ThioHis-TOPO1. Resin bound proteins were examined by western blotting using anti-Thio (upper panel) or anti-GST (lower panel) antibodies. C. Interaction of GST-YB-1 deletion mutants with TOPO1 in nuclear lysis solutions of PC-3 cells. Approximately 500 μg of each GST fusion protein was immobilized on 15 μl of glutathione-Sepharose 4B, and the resin was incubated with 500 μg of PC-3 nuclear lysis. Resin bound proteins were western blotted and the membrane probed using TOPO1 (upper panel) or anti-GST (lower panel) antibodies.