Figure 4.
Figure 4. Unscheduled activation of Cdk1 in PP4c−/− MEF cells. (A) Aberrant phosphorylation at T219 of NDEL1 in PP4c−/− MEF cells (48 h after infection of PP4ccko/cko MEF cells with adeno-Cre). Uninfected PP4ccko/cko MEF cells were used as controls. (top) T219-phosphorylated NDEL1 was detected by using a phospho-T219–specific monoclonal antibody (N219TP). Arrowheads indicate aberrant N219TP staining and γ-tubulin of PP4c−/− MEF cells in interphase. (middle) Statistical analysis of N219TP-positive cells in interphase (*, P < 0.001; one example of three independent experiments; n = 100). (bottom) Western blotting pattern using a phospho-T219–specific monoclonal antibody. An anti-NDEL1 antibody was used for a control. Note that phosphorylated NDEL1 displayed lower mobility. (B) Unscheduled phosphorylation of cyclin B1 of PP4c−/− MEF cells in interphase 48 h after infection of PP4ccko/cko MEF cells with adeno-Cre. Uninfected PP4ccko/cko MEF cells were used as controls. (top) Antiphospho–cyclin B1 (phospho-S123) staining in PP4c−/− MEF cells. (middle) Statistical analysis of phospho-S123–positive cells (*, P < 0.001; one example of three independent experiments; n = 100). Error bars represent SEM. (bottom) Western blotting pattern using an antiphospho–cyclin B1–specific monoclonal antibody. An anti–cyclin B1 antibody was used for a control. (A and B) Arrowheads indicate the positions of centrosomes. (C) Histone H1 kinase assay. GFP-PACT-Cdk1 and Red-Cre plasmids were transfected to PP4c+/+ or PP4ccko/cko MEF cells. GFP-PACT-Cdk1 was precipitated by an anti-GFP antibody 48 h after transfection followed by a kinase assay using histone H1 as a substrate. Note that GFP-PACT-Cdk1 extracted from PP4c−/− MEF cells displayed higher kinase activity. (D) Flow cytometric analysis of PP4c−/− MEF cells (top) and immunostaining pattern using an antiphosphohistone H3 antibody (bottom) 48 h after infection of PP4ccko/cko MEF cells with adeno-Cre. Uninfected PP4ccko/cko MEF cells were used as controls. Although flow cytometry revealed the enrichment of cell populations in 2n and 4c, phosphohistone H3–positive cells were rarely detected, suggesting that most of the population of PP4c−/− MEF cells was arrested in G2 before entering into prophase. One example of three independent experiments is shown. Bars, 10 μm.

Unscheduled activation of Cdk1 in PP4c−/− MEF cells. (A) Aberrant phosphorylation at T219 of NDEL1 in PP4c−/− MEF cells (48 h after infection of PP4ccko/cko MEF cells with adeno-Cre). Uninfected PP4ccko/cko MEF cells were used as controls. (top) T219-phosphorylated NDEL1 was detected by using a phospho-T219–specific monoclonal antibody (N219TP). Arrowheads indicate aberrant N219TP staining and γ-tubulin of PP4c−/− MEF cells in interphase. (middle) Statistical analysis of N219TP-positive cells in interphase (*, P < 0.001; one example of three independent experiments; n = 100). (bottom) Western blotting pattern using a phospho-T219–specific monoclonal antibody. An anti-NDEL1 antibody was used for a control. Note that phosphorylated NDEL1 displayed lower mobility. (B) Unscheduled phosphorylation of cyclin B1 of PP4c−/− MEF cells in interphase 48 h after infection of PP4ccko/cko MEF cells with adeno-Cre. Uninfected PP4ccko/cko MEF cells were used as controls. (top) Antiphospho–cyclin B1 (phospho-S123) staining in PP4c−/− MEF cells. (middle) Statistical analysis of phospho-S123–positive cells (*, P < 0.001; one example of three independent experiments; n = 100). Error bars represent SEM. (bottom) Western blotting pattern using an antiphospho–cyclin B1–specific monoclonal antibody. An anti–cyclin B1 antibody was used for a control. (A and B) Arrowheads indicate the positions of centrosomes. (C) Histone H1 kinase assay. GFP-PACT-Cdk1 and Red-Cre plasmids were transfected to PP4c+/+ or PP4ccko/cko MEF cells. GFP-PACT-Cdk1 was precipitated by an anti-GFP antibody 48 h after transfection followed by a kinase assay using histone H1 as a substrate. Note that GFP-PACT-Cdk1 extracted from PP4c−/− MEF cells displayed higher kinase activity. (D) Flow cytometric analysis of PP4c−/− MEF cells (top) and immunostaining pattern using an antiphosphohistone H3 antibody (bottom) 48 h after infection of PP4ccko/cko MEF cells with adeno-Cre. Uninfected PP4ccko/cko MEF cells were used as controls. Although flow cytometry revealed the enrichment of cell populations in 2n and 4c, phosphohistone H3–positive cells were rarely detected, suggesting that most of the population of PP4c−/− MEF cells was arrested in G2 before entering into prophase. One example of three independent experiments is shown. Bars, 10 μm.

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