Fig. 4
Nuclear activity of Polo activates Cdc25 in prophase. a GFP-Cdc25 is rapidly excluded from the nucleus in prophase. The green box indicates the time of initial detection of GFP-Cdc25 in the cytoplasm before NEB set as T 0. Bar: 5 μm. In this experiment, cells were mock-treated with medium containing DMSO as a control for Supplementary Fig. 6b. b, c The nuclear exclusion of GFP-Cdc25 coincides with the nuclear import of Polo-RFP. Bar: 5 μm. (n = 10, error bars: SD). d Cells were transfected and treated with BI 2536 as indicated and were fractionated into cytoplasmic (C) and nuclear (N) fractions analyzed by western blots. The position of Cdc25 and its phosphorylated forms are indicated by arrows. MEK and Histone H3 are, respectively, cytoplasmic and nuclear proteins probed as controls. e Cdc25 phosphorylation is regulated by Polo kinase in the nucleus. D-Mel2 cells were transiently transfected as indicated. BI 2536 was added at 300 nM. Lysates were analyzed by western blotting. Inhibitory phosphorylation of Cdk1 was monitored using anti-pY15 antibodies. f Polo promotes Cdc25 activation in the nucleus. After transfection with the indicated constructs, Myc-Cdc25 (WT or phosphatase-dead) was immunoprecipitated and tested in a phosphatase assay. A representative experiment is shown with duplicate readings. AU arbitrary units. Error bars: SD. g The interaction between Polo and Cdc25 is regulated by Cdk1. Cells were transfected with Myc-Cdc25 and Polo-GFP (WT or NLS7A) plasmids, and treated or not with the Cdk1 inhibitor RO 3306 (10 μM). Cell lysates were subjected to immunoprecipitation with anti-GFP antibodies and immunoblotted with the indicated antibodies
