Fig. 3. Endos regulates Twine, Polo and MPM2 phosphoepitopes. (A) Anti-β-gal western blot, showing reduced expression of Twine::β-gal in endos⁰⁰⁰⁰³ and endos⁰⁰⁰⁰³ elgi¹ stage 14 oocytes (30 stage 14 oocytes per lane). (B) X-gal staining (blue) of control, endos⁰⁰⁰⁰³, elgi¹ and endos⁰⁰⁰⁰³ elgi¹ oocytes reflecting Twine::β-gal levels at stages 13 and 14. Scale bar: 100 µm. (C) Polo western analysis of control, twine¹, endos⁰⁰⁰⁰³, elgi¹ and endos⁰⁰⁰⁰³ elgi¹ stage 14 oocytes showing a strong reduction of Polo levels in endos⁰⁰⁰⁰³ and endos⁰⁰⁰⁰³ elgi¹ (50 stage 14 oocytes per lane). (D) Cyclin B western analysis showing normal expression in endos⁰⁰⁰⁰³ and elgi¹ mutants, and slightly elevated expression in twine¹ mutants (30 stage 14 oocytes per lane). (E) In vitro Cdk1 kinase assay using anti-Cdk1 or anti-Cyclin B immunoprecipitates (IP) from control, twine¹, endos⁰⁰⁰⁰³ and elgi¹ stage 14 extracts. Mock immunoprecipitates were performed without antibodies. Immunoprecipitates were either immunoblotted using anti-Cdk1 or anti-Cyclin B antibodies, or subjected to a kinase assay using [³²P]ATP and histone H1 as substrate. (F) Western blotting using MPM2 antibodies. Wild-type and elgi¹ oocytes show high MPM2 levels, whereas endos⁰⁰⁰⁰³, twine¹ and endos⁰⁰⁰⁰³ elgi¹ oocytes have drastically reduced MPM2 phosphoepitopes (50 stage 14 oocytes per lane). Actin was used as a control.