Fig. 5. Effect on Barren and SMC4 of CAP-D2 depletion. (A) Immunoblotting of control and CAP-D2-depleted extracts with Barren and SMC4 antibodies demonstrates that Barren levels are reduced while SMC4 levels appear largely unaffected after loss of CAP-D2. -tubulin was used as a loading control. C, control dsRNA; –, no RNA; D, CAP-D2 dsRNA. (B,C) Cells from both control (B) and CAP-D2 RNAi (C) experiments were cytospun onto poly-L-lysine slides 72 hours after treatment and stained for Barren (red), Cyclin B (green) and DNA (blue). Barren is absent from mitotic chromosomes and localised diffusely in the cytoplasm after CAP-D2 RNAi. (D,E) Control (D) and CAP-D2 RNAi (E) cells were cytospun onto poly-L-lysine slides 65 hours after treatment, extracted during fixation, and stained for SMC4 (red), CAP-D2 (green) and DNA (blue). While cells have varying levels of SMC4 on chromatin, any axial localisation of SMC4 is lost in the CAP-D2-depleted cells. Scale bar: 10 µm.