14-3-3ε collaborates with Polo to promote the cytoplasmic localization of Gwl. (A) Gwl is copurified with 14-3-3 proteins in a GST pulldown. An extract from D-Mel cells expressing Gwl-Myc was incubated with GST fusions proteins on Sepharose. (B) Gwl is copurified with 14-3-3 proteins from cells in culture. Cells coexpressing Gwl-Myc and Protein A fusion proteins as indicated were used in PrA affinity purifications. (C and D) Overexpression of 14-3-3ε enhances the cytoplasmic localization of Gwl induced by a gain of Polo function. (C) Examples of cells analyzed by immunofluorescence. Bar, 10 µm. (D) Quantification. For each condition, the cytoplasmic and nuclear fluorescence of Gwl-Myc was measured for multiple interphase cells taken randomly. Error bars: SEM. Asterisks: P < 0.001 after Student’s t test. (E) Polo consensus sites but not Cdk consensus sites in the central region of Gwl are required for its interaction with 14-3-3ε. Cells expressing the indicated proteins were used in PrA affinity purifications followed by Western blots. (F) Treatment of cells with a Polo inhibitor (BI2536, 200 nM) but not a Cdk1 inhibitor (RO-3306, 10 µM) abrogated the interaction of Gwl-Myc with 14-3-3ε in a GST pulldown.