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Figure 5


Fig. 5. Aurora B phosphorylates condensin I regulatory subunits in vitro and in vivo. (A-C) Condensin was purified by immunoprecipitation from EGFP-FLAG-kleisin-{gamma} (A), EGFP-FLAG-Cap-D2 (B) (both condensin I) and EGFP-FLAG-kleisin-beta (C) (condensin II) cell lines. Immunoprecipitates were incubated either with or without recombinant Aurora B and [{gamma}-32P]ATP in vitro, resolved on a gel, silver stained (Silver) and exposed (32P). The identity of the bands was determined by mass spectrometry. (D) Condensin I (EGFP-FLAG-kleisin-{gamma}, first panel) and condensin II (EGFP-FLAG-kleisin-beta, second panel) were immunoprecipitated either from interphase (hydroxyurea arrested; HU) or mitotic cells (Nocodazole arrested; Noc) and analysed for electrophoretic mobility shifts that are sensitive to pre-treatment of the HeLa cells with Hesperadin (100 nM for 1 hour) or to treatment of the immunoprecipitates with {lambda}-phosphatase. Total mitotic extracts (ME) were probed with antibodies against H3S10ph to monitor Aurora B activity and against cyclin B to verify that cells were in mitosis.





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