Aurora B activity at a distance depends on phosphorylation of INCENP. (A) HeLa cells were transfected with either CB^DBD-INCENP–mCherry or CB^DBD-INCENP^TSS/AAA–mCherry, together with an Aurora B phosphorylation sensor targeted either to centromeres, chromatin, or kinetochores. Cells were also treated with or without Borealin siRNA and imaged live. The normalized YFP/CFP emission ratio or YFP/TFP for the kinetochore-targeted sensor was calculated as described in Fig. 1 C and averaged over three independent experiments. (B and C) Cells were transfected with the indicated constructs with or without INCENP siRNA as indicated and then were fixed and stained for phospho-Dsn1 (pDsn1) Ser100 or phospho-H3 (pHH3) Ser10. Representative images are shown (B), and normalized phospho-Dsn1 or phospho-H3 staining was quantified and averaged over three independent experiments (C). (D and E) Cells transfected as described in B and C were fixed and analyzed for cold-stable microtubules. (D) Images are maximum intensity projections of confocal stacks; the insets are optical sections showing individual kinetochores on the right. (E) The percentage of kinetochores with cold-stable microtubules from multiple cells was averaged over three independent experiments. Bars: (B and D) 5 µm; (D, insets) 2.5 µm.