Fig. 6: Phosphorylation of DAB2IP at its Thr531 and Thr546 sites activates the PLK1-Mps1 signal pathway and inhibits Cdc20 ubiquitylation in prometaphase. | Oncogene

Fig. 6: Phosphorylation of DAB2IP at its Thr531 and Thr546 sites activates the PLK1-Mps1 signal pathway and inhibits Cdc20 ubiquitylation in prometaphase.

From: Mitotic phosphorylation of tumor suppressor DAB2IP maintains spindle assembly checkpoint and chromosomal stability through activating PLK1-Mps1 signal pathway and stabilizing mitotic checkpoint complex

Fig. 6

A Flag-tagged-DAB2IP, -DAB2IP 2A (T531A/T546A), -DAB2IP 2D (T531D/T546D), empty vector and HA-PLK1 were transfected into HeLa cells. The cells were then treated with 50 ng/ml nocodazole for 16 h and mitotic cells lysates were immunoprecipitated with anti-Flag antibody; the HA signal was examined by immunoblotting. B Immunoblotting analysis levels of PLK1-pT210, PLK1, Mps1, BubR1 in mitotically arrested or asynchronous PC3 cells with siRNA-mediated DAB2IP suppression and overexpression of siRNA-resistant DAB2IP (rWT), siRNA-resistant DAB2IP 2A (r2A), and siRNA-resistant DAB2IP 2D (r2D) constructs. C PC3 cells with siRNA-mediated DAB2IP suppression and overexpression of siRNA-resistant DAB2IP (rWT), siRNA-resistant DAB2IP 2A (r2A), and siRNA-resistant DAB2IP 2D (r2D) constructs were arrested in mitosis by nocodazole, and shake-off cell lysates were immunoprecipitated by anti-Mps1 antibody. The phosphorylation of Mps1 on its Thr676 site and the amount of Mps1 protein were detected. D Flag-tagged-DAB2IP, -DAB2IP 2A (T531A/T546A), -DAB2IP 2D (T531D/T546D), empty vector and Myc-Cdc20 were transfected into HeLa cells. The cells were then treated with 50 ng/ml nocodazole for 16 h and mitotic cells lysates were immunoprecipitated with anti-Flag antibody; the Myc signal was examined by immunoblotting. E Myc-Cdc20 and HA-Ubi were co-transfected with Flag-DAB2IP, Flag-DAB2IP 2A (T531A/T546A), Flag-DAB2IP 2D (T531D/T546D) or empty vector in HeLa cells. Cells were harvested at 6 h after MG132 (10 μM) treatment. Cdc20 was immunoprecipitated by using anti-Myc antibody. Co-IP products were analyzed by immunoblotting using anti-HA and anti-Myc antibodies. Equal loadings of whole cell lysates were subjected to immunoblotting by anti-Flag and anti-HA antibodies. F The intensity of ubiquitinated proteins was normalized to VC transfected cells, then graphed for four independent experiments (n = 4; ***P < 0.001 as compared with VC transfected cells.).

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