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Fig. 3 | Molecular Cancer

Fig. 3

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

Fig. 3

c-Src phosphorylates DMAP1 at Tyr246 and disrupts Bub3/DMAP1 complex formation. In a-e, immunoblotting analyses were performed using the indicated antibodies. Data represent 1 out of 3 experiments. In a, c and d, cells were synchronized in interphase (I) by thymidine double block (2 mM) or were synchronized in mitosis (M) by nocodazole (200 nM) treatment for 16 h after releasing thymidine double block for 8 h. In a-e, data represent 1 out of 3 experiments. a, PANC-1 or SW1990 cells were synchronized in interphase or mitosis. Cellular extracts were subjected to immunoprecipitation with an anti-Src antibody. b, In vitro phosphorylation analyses were performed by mixing the purified active c-Src with the indicated purified GST-DMAP1 proteins in the presence of [γ-32P]ATP. Tyr246 of DMAP1 is evolutionarily conserved in the indicated species (b, left panel). c, Cells synchronized in mitosis were expressed with indicated Flag-DMAP1. Cellular extracts were subjected to immunoprecipitation with an anti-Flag antibody Cells. d, The indicated purified GST-DMAP1 protein was mixed with mitotic extracts from PANC-1 cells in the presence or absence of c-Src. GST pull down analyses were performed. e, Purified His-Bub3 protein with or without phosphorylation by c-Src was mixed with GST-DMAP1 purified protein with or without p38. GST pull down analyses were performed

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