Fig. 7. Localisation of DRAD21 in DmSMC4-depleted Drosophila S2 cells. (A) Immunolocalisation of DmSMC4 and DRAD21 in control cells. In prometaphase cells, DmSMC4 and DRAD21 do not appear to co-localise. DRAD21 is always confined to the centromeric region between the axial structure stained by DmSMC4. During anaphase while DmSMC4 localises throughout sister chromatids, DRAD21 is not detected. (B) After depletion of DmSMC4, DRAD21 is still highly abundant in prophase chromosomes but during prometaphase it is restricted to defined regions of the condensed chromosomes. In anaphase, DRAD21 is never detected either on the chromatids or the chromatin bridges. Scale bars: 5 µm. (C) Western blot of total protein extracts from control and DmSMC4-depleted cells after 96 hours show that depletion of DmSMC4 does not affect the overall levels of DRAD21 but Barren levels are reduced compared to control extracts. (D) Analysis of protein extracts from immunoprecipitated mitotic chromatin of either control or DmSMC4-depleted cells and the corresponding supernatants. In chromatin extracts from control cells DmSMC4, DRAD21 and Barren can be easily detected while in extracts from dsRNAi-treated cells neither DmSMC4 nor Barren are detected and the level of DRAD21 is reduced compared to controls. Phosphohistone H3 was used as a loading control. The corresponding supernatants are shown below. DmSMC4 is not detected by immunoblotting and the levels of DRAD21 are not different from controls. However, note that depletion of DmSMC4 causes a significant decrease in the level of Barren present in the supernatant fraction. -tubulin was used as loading control.