Aurora-B kinase is expressed in EnM MKs. (A) RT-PCR assay for Aurora-B kinase using RNA isolated from polyploid murine MKs and unsynchronized BaF3/Mpl cells. Two independent primer pairs were designed to yield products of about 300 base pair (bp). (B) Western blot comparing Aurora-B kinase expression in polyploid murine MKs and diploid cells. Gradient-purified MKs compared to unsynchronized BaF3/Mpl cells. Equivalent amounts of total cell protein were loaded in each lane. Aurora-B kinase is 41 kDa. (C) Kinase assay comparing Aurora-B kinase activity in polyploid murine MKs and diploid cells. Aurora-B kinase was immunoprecipitated from mature MK or BaF3/Mpl lysates, and kinase activity was assayed using purified histone H3 as a substrate. Phosphorylation was detected by blotting with phospho-specific histone H3 antibody. Blots were reprobed with antibody to histone H3. Lanes 1 and 2 contain no substrate, lane 3 contains purified histone H3 alone, lane 4 is empty, and lanes 5 and 6 show the kinase assay of MK and BaF3/Mpl immunoprecipitates against the purified histone H3 (19 kDa).