Fig. 1

Bub3 interacts with DMAP1 during mitotic arrest. In A-F, immunoblotting analyses were performed using the indicated antibodies; Data represent 1 out of 3 experiments. a, HPDE cells expressing Flag-Bub3 were synchronized in interphase by thymidine double block (2 mM) or were synchronized in mitosis by nocodazole (200 nM) treatment for 16 h after releasing thymidine double block for 8 h (left panel), then cells were extracted and subjected to immunoprecipitation with an anti-Flag antibody. The precipitates from immunoprecipitated-Flag-bub3 were washed by Flag peptides and then were analyzed by coomassie brilliant blue staining and immunoblotting (right panel). b, HPDE cells were synchronized by thymidine double block (2 mM) and were released for 8 h, followed by nocodazole (200 nM) treatment for 16 h, cellular extracts were subjected to immunoprecipitation with an anti-Bub3 antibody. c, HPDE cells were transfected with or without DMAP1 siRNA. Cellular extracts were subjected to immunoprecipitation with an anti-Bub3 antibody. d, HPDE and PANC-1 cells were synchronized by thymidine double block (2 mM) and were released for 8 h, followed by nocodazole (200 nM) treatment for 16 h (left panel). PANC-1 cells were treated with SU6656 (shown as ‘SU’) (10 μM) for 1 h post Nocodazole treatment for 16 h. Cellular extracts were subjected to immunoprecipitation with an anti-Bub3 antibody. e, HPDE cells overexpressed with Src Y527F were synchronized by thymidine double block (2 mM) and were released for 8 h, followed by nocodazole (200 nM) treatment for 16 h (left panel). Cellular extracts subjected to immunoprecipitation with an anti-Bub3 antibody. f, HPDE cells synchronized by thymidine double block (2 mM) were released for 8 h, followed by nocodazole (200 nM) treatment for 16 h (left panel). Cells expressing HA-DMAP1was transfected with plasmids expressing indicated length of Flag-Bub3. Cellular extracts were subjected to immunoprecipitation with an anti-Flag antibody