JCS01033 Supplemental Figures

Files in this Data Supplement:

• Figure S1 -

  Fig. S1. Characterization of the polyclonal antibodies made against Bub1, Mad2, Bub3 and BubR1. Western blots using pre-immune sera (PI) show that they do not recognise antigens of the expected molecular mass in S2 cell extracts. Western blots using immune sera (IS) against Bub1, Mad2, Bub3 and BubR1 show them to contain specific antibodies that detect bands of the predicted molecular mass: 125 kDa, 25 kDa, 37 KDa and 165 kDa, respectively. Affinity-purification of the antibodies against Bub1, Mad2 and Bub3 abolishes recognition of cross-reacting bands.

• Figure S2 -

  Fig. S2. Drosophila S2 culture cells delay mitotic progression in response to microtubule poisons. (A) Quantification of cell growth over time in control cells, cells treated with low doses of taxol (10 nM) and cells incubated with colchicine (30 mM). Cell number remains constant after addition of colchicine, and incubation with taxol slightly delays the culture doubling time. (B) Mitotic index of the different cultures at different times. Mitotic index increases moderately after taxol treatment and strongly after colchicine treatment. After 12 hours the mitotic indexes start decreasing in both cases. (C) Quantification of cell viability in the different cultures.

• Figure S3 -

  Fig. S3. Bub3 is recruited to phosphorylated but not to unphosphorylated kinetochores. Cells were initially lysed in the absence of microcystin to become dephosphorylated, treated with ATP plus microcystin to rephosphorylate kinetochore proteins, incubated with a complete (M ext) or Bub3-depleted (M ext –Bub3) S2 mitotic extract, fixed and processed for immunofluorescence. DNA is shown in blue. Exogenously added Bub3 localizes strongly after incubation with complete mitotic extract while little or no localization is observed after incubation with Bub3-depleted extract. Scale bars: 5 mm.