FIGURE 2.

Topo IIβ-SP120 complex contains RNA. A, HEK293 cells were co-transfected with pFLAG-topo 2b and pMyc-SP120 and the whole cell extract was used for immunoprecipitation by anti-FLAG antibody. The expressed proteins in IP were detected by Western blotting with anti-FLAG (left panel) or anti-Myc (right panel) antibodies. B, FLAG-tagged proteins were expressed separately in HEK cells and immunopurified in high-salt conditions. These preparations were free from cross-contamination as shown by the stained gel (arrowheads). C, immunopurified FLAG-topo IIβ and FLAG-SP120 were mixed in 0.1 m NaCl and immunoprecipitated with anti-topo IIβ peptide antibody. The protein complex formed on beads was treated with increasing concentrations of NaCl as indicated. Proteins remaining on beads were separated by SDS-PAGE and detected by staining (upper panels). Relative amounts of SP120 are plotted in the lower graph. D, high-salt (0.42 m NaCl) cell extracts were treated or untreated with RNase A, and the proteins were immunopurified. Amounts of RNA associated with proteins were quantified by RiboGreen. RNA per 100 ng of protein is shown in the graph.