FIGURE 3.

The C-terminal domain of SP120 with tightly bound RNA is involved in the complex formation. A, domain structure of SP120 and its variants used for the analysis. All recombinant proteins had Myc tag on the N terminus. Numbers in parentheses stand for amino acid residue numbers. SAF-box and RGG-box in SP120 have been ascribed as essential motifs for the binding of DNA and RNA, respectively. NLS, nuclear localization signal. B, HEK cells were co-transfected with FLAG-topo IIβ and Myc-SP120 (WT, ΔC, or ΔN). The expressed proteins were detected by anti-tag antibodies (left panel). Cell extracts were immunoprecipitated with anti-FLAG antibody and subjected to Western blotting with anti-tag antibodies (right panel). C, tagged proteins were expressed separately in HEK cells and tag-purified in high-salt extract. Myc-SP120 extracts were either treated (+) or untreated (−) with RNase A. SP120 proteins (WT and C187) fixed on beads through anti-Myc antibody were incubated with FLAG-topo IIβ free in solution. Proteins bound on the beads were detected by Western blotting with anti-tag antibodies. The asterisk indicates the light chain of the antibody used for IP.