Endos-RFP localization to the nuclear/spindle area is independent from interaction with PP2A-Tws. (A) IF against endogenous Endos reveals specific staining in the nuclear area in prometaphase. Scale bar: 20 µm. (B) Endos-RFP but not Endos^S68A-RFP or Endos^Δ41–80-RFP rescues the development of Endos mutant flies. Transgenes were under the UASp promoter and driven ubiquitously by Ubi-Gal4. Values shown correspond to percentages of eclosed endos¹/Df pupae (Tb⁺) relative to the expected number of endos¹/Df pupae calculated from the total number of eclosed pupae. The observed rescue exceeds the expected rescue because of higher stochastic mortality of the other genotypes caused by balancer chromosomes. Values are averages of three independent experiments in which between 193 and 325 eclosed pupae were scored for each cross. Error bars: SD. ****, P < 0.0001. (C–E) Time-lapse imaging of embryos expressing GFP-Tws and variants of Endos-RFP. (C) Endos^S68D-RFP (gain of interaction with PP2A-Tws). (D) Endos^Δ41–60-RFP (loss of pS68-independent interaction with PP2A-Tws). (E) Endos^Δ41–80-RFP (complete loss of interaction with PP2A-Tws). Images from multiple z-steps were combined in an average intensity projection. (F) Validation of the phosphospecific antibody against pS68-Endos. Cells were transfected and treated as indicated and analyzed by WB. Ab, antibody. Scale bars: 5 µm (C–E).