Fig. 5: Cdk1 mediates phosphorylation of DAB2IP on its Thr531 and Thr546 sites in mitosis. | Oncogene

Fig. 5: Cdk1 mediates phosphorylation of DAB2IP on its Thr531 and Thr546 sites in mitosis.

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. 5

A Thymidine-synchronized PC3 cells were released into normal medium containing nocodazole (50 ng/ml) and harvested at the indicated time points after release. The expression of DAB2IP, Cdc27, cyclin B1, securin, and phosphorylation of histone 3 on Ser10 were determined by using immunoblotting. B Mitotically arrested PC3 cells were released for cell cycle reentry and harvested at the indicated time points after release. The expression of DAB2IP, Cdc27, cyclin B1, securin, and phosphorylation of histone 3 on Ser10 were determined by using immunoblotting. C Mitotically arrested (nocodazole- or paclitaxel-induced) and asynchronous PC3 cells lysates were treated with or without λ-PPase. The expression pattern of DAB2IP was presented by using immunoblotting. D DAB2IP-proficent PC3 and C4-2 D2 cells were transfected with siRNA against Cdk1 or control siRNA, and 24 h after transfection, cells were treated with nocodazole for an additional 16 h. The expression of DAB2IP, Cdk1, cyclin B1 and Actin in mitotically arrested and asynchronous cells was determined by immunoblotting. E Sequence alignment of the DAB2IP proteins from different species (Homo sapiens, Mus musculus, Rattus norvegicus, Pan troglodytes, and Canis lupus) around Thr531 and Thr546. F Flag-tagged wild-type DAB2IP, DAB2IP T531A, DAB2IP T546A, DAB2IP 2A (T531A/T546A) and empty vector were expressed in HeLa cells, and HeLa cells were synchronized by nocodazole. The wild-type form of DAB2IP and different mutants were immunoprecipitated by anti-Flag (M2) antibody from mitotically arrested cell lysates. Phosphorylations on DAB2IP and its mutants were detected by immunoblotting using an antibody that recognizes Cdk targeting TP motifs (p-TP). The expression of Flag-tagged protein was also determined. G Flag-tagged wild-type DAB2IP, DAB2IP 2A (T531A/T546A) and empty vector were expressed in HeLa cells, and HeLa cells were synchronized by nocodazole. The wild-type form of DAB2IP and the 2A mutant were immunoprecipitated by anti-Flag (M2) antibody from mitotically arrested cells lysates. The phosphorylation of DAB2IP on its Thr531 site and the expression of DAB2IP were detected.

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