Fig. 7. Bub1 is phosphorylated in response to spindle damage. (A) HeLa cells synchronised by a double thymidine block analysed by flow cytometry (top panel) and western blotting using 4B12 (anti-Bub1, bottom left panel) and 5F9 (anti-BubR1, bottom right panel) at various times following release from G₁/S, shown in hours. The anti-tubulin antibody TAT-1 (Woods et al., 1989) was used to monitor protein loading. Between 9 and 12 hours after release, the majority of the cells had completed mitosis and returned to G₁. In the presence of nocodazole, both Bub1 and BubR1 exhibit slower migrating forms. (B) Proteins from mitotic (M) HeLa cells treated with phosphatase (+) or CIP (+C), as indicated, then blotted for Bub1 using SB1.3 or BubR1 using 5F9. Phosphatase treatment results in the disappearance of the slower migrating forms indicating that they are phosphorylated forms. (C) Mitotic L929 (M, upper panel) or mitotic HeLa cells (M, lower two panels) were treated with 0.2 µg/ml nocodazole (N) or 10 µM taxol (T) for the times indicated in minutes then blotted for Bub1 using 4B12 or BubR1 using 5F9. Phosphorylated Bub1 is only detectable in response to spindle damage but BubR1 is phosphorylated in the absence of spindle toxins. (D) Bub1 (lanes 1 and 4) and BubR1 (lanes 3 and 6) were immunoprecipitated from nocodazole-arrested TA-HeLa cells using the 4B12 and 5F9 antibodies, respectively. The immunoprecipitates were then analysed by western blotting using 4B12 (anti-Bub1, right panel) and 5F9 (anti-BubR1, left panel). The lower panel shows that when the two forms of BubR1 are well resolved, it becomes apparent that the phosphorylated form of BubR1 preferentially immunoprecipitates with Bub1.