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Fig. 2 | Molecular Cancer

Fig. 2

From: C-Src confers resistance to mitotic stress through inhibition DMAP1/Bub3 complex formation in pancreatic cancer

Fig. 2

p38 phosphorylates Bub3 at Ser211 and promotes Bub3/DMAP1interaction. In a-h, immunoblotting analyses were performed using the indicated antibodies. Data represent 1 out of 3 experiments. In a-d and h, cells were synchronized in interphase (I) by thymidine (2 mM) double block or were synchronized in mitosis (M) by nocodazole (200 nM) treatment for 16 h after releasing thymidine double block for 8 h. In a-h, data represent 1 out of 3 experiments. a, HPDE cells were synchronized in interphase or mitosis. Cellular extracts were subjected to immunoprecipitation with an anti-Bub3 antibody and the immunoprecipitates were treated with or without CIP (10 units). b, HPDE cells synchronized in interphase or mitosis were treated with Compound C (10 μM), SP600125 (20 μM) and SB203580 (25 μM) for 1 h, after Nocodazole treatment for 16 h. c, PANC-1 or SW1990 cells were synchronized in interphase or mitosis. Cells were treated with SB203580 (25 μM) and SU6656 (shown as ‘SU’) (10 μM) for 1 h, after Nocodazole treatment for 16 h. Cellular extracts were subjected to immunoprecipitation with an anti-Bub3 antibody. d, HPDE or PANC-1 cells were synchronized in interphase or mitosis. Cellular extracts were subjected to immunoprecipitation with an anti-p38 antibody. e and f, In vitro phosphorylation analyses were performed by mixing the purified active p38 with the indicated purified GST-Bub3 proteins in the presence of [γ-32P]ATP. Ser211 of Bub3 is evolutionarily conserved in the indicated species (f, left panel). g, The indicated purified His-Bub3 protein was mixed with GST-DMAP1 purified protein with or without p38. GST pull down analyses were performed. h, HPDE cells synchronized in mitosis were expressed with indicated Flag-Bub3. Cellular extracts were subjected to immunoprecipitation with an anti-Flag antibody

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