Quantitation of aurora B kinase activity in INCENP mutants. (A) Estimation of aurora B activity by immunoblotting. Asynchronous cells were harvested after treatment with doxycycline for 28 h or with 2 µM ZM447439 for 5 h (Fig. S1 C), and lysates were subjected to immunoblotting with the indicated antibodies. α-Tubulin and haspin kinase substrate H3T3ph are shown as controls. White lines indicate that intervening lanes have been spliced out. (B) Measurement of H3S10ph levels in the same samples using Odyssey. (C) Ratio of aurora B protein levels versus the loading control α-tubulin for each sample as measured using Odyssey. (D) Mitotic index of each cell line at the time of harvesting. (E) Diagram showing the relative levels of aurora B activity based on the level of H3S10ph measured using the Odyssey assay. WT, wild type. Error bars indicate SD.