Fig. 5. Determination of the level of Topo II associated with chromatin in DmSMC4-depleted cells. (A) The levels of Topo II in total protein extracts from control or DmSMC4-depleted cells after 96 hours show a 19.5% reduction. (B) To determine whether this reduction corresponds to the chromosomal-associated Topo II, increasing amounts of mitotic chromatin extracts from control and DmSMC4-depleted cells immunoprecipitated with anti-phosphohistone H3 antibodies or the remaining supernatant were separated by SDS-PAGE and immunoblotted. Membranes were probed with antibodies against DmSMC4 and Topo II. Histone H3 was used as a loading control for immunoprecipitated chromatin and -tubulin for the supernatant. While DmSMC4 and Topo II are clearly found in the control extracts, in the chromatin from DmSMC4-depleted cells DmSCM4 is absent and the levels of Topo II are not significantly different from controls extracts. In the corresponding supernatant fractions, DmSMC4 and Topo II are easily detected in the control cells. However, after DmSMC4 dsRNAi treatment, DmSMC4 is not detected and Topo II is reduced.