Figure 2. Full Bub1 activation is mitotic specific and requires the kinase extension domain.

(a) Evolutionary conservation of Bub1 T589 and S679. (b) Bub1 deletion mutants were expressed in HeLa cells depleted of endogenous Bub1. Mitotic cells were stained with Hoechst (Blue in merge), anti-MYC (green) and anti-pT589 (red). Scale bar, 10 μM. Quantitation of pT589 signal relative to CREST at kinetochores (mean±s.e.) from a minimum of ten cells per condition is indicated in the right-most panel. (c) Cells were transfected with Bub1 mutants as in b and enriched in mitosis by nocodazole treatment. Anti-pT679 (upper half) and anti-pT589 (bottom half) western blottings were performed with MYC-Bub1 immunoprecipitated from equalized lysates. Anti-MYC blotting (second and fourth panels) reveals equal loading. (d) MYC-Bub1 was immunoprecipiated from HeLa cells stably expressing MYC-Bub1-WT arrested in G1/S or mitosis by thymidine (THY) or nocodazole (NOC) treatment, respectively, and blotted with anti-pT679 antibodies (upper panel) or stripped and reprobed with anti-MYC. (e) Western blottings of histones purified from thymidine- and nocodazole-arrested cells with anti-H2A-pT120 (upper panel) and anti-H2A (lower panel) antibodies. (f,g) U2OS cells expressing a 256-copy array of the lac operator were transfected with a LacI-GFP, 3XMYC-LacI-Bub1-WT or KD. Fixed cells were stained with Hoechst (blue), anti-MYC or GFP in the control (green) and either anti-H2A-pT120 (red, f) or anti-Sgo1 (red, g). The overlap between the MYC and H2A-pT120 or Sgo1 is shown in the panel on the right of each figure. Error bars represent s.e. Scale bar, 5 μM.