The Nesd N-terminal domain is required for midbody localization and interaction with centralspindlin. (A) Western blot analysis of Nesd protein levels in Drosophila S2 cells treated for 72 h with either GFP (control) or nesd dsRNA. The arrow marks the band corresponding to the Nesd protein, whereas the asterisk marks a nonspecific band used as a loading control. (B) Drosophila S2 cells were fixed and stained to detect Nesd, tubulin (Tub), and DNA. (C) Drosophila S2 cells were fixed and stained to detect Nesd, Pav-KLP (Pav), and DNA (blue in the merged image). The punctate staining in the red channel of the early telophase cell is not specific because it is not eliminated after nesd RNAi (Fig. S1 B). Bars, 5 µm. (B and C) Insets show magnifications of the midbody. (D) Drosophila S2 cells stably expressing Nesd::GFP were fixed and stained to detect GFP, tubulin, and DNA. The arrowhead marks Nesd localization to the midbody ring, whereas the arrow indicates a midbody remnant. (E) Drosophila cells stably expressing two Nesd fragments (1–292 and 293–602) tagged with GFP were fixed and stained to detect GFP, tubulin, and DNA. (B, D, and E) Bars, 10 µm. (F) Schematic representation of the midbody localization and interaction with RacGAP50C of several Nesd fragments. The positions of the NHD and PLD are indicated. Nesd fragments were tagged at either the N or C terminus with GFP, and their localizations were then analyzed in S2 cells. C-terminal myc-tagged Nesd fragments were used to test the interaction with RacGAP50C. (G) Drosophila S2 cells stably expressing RacGAP::PrA were transfected with the indicated myc-tagged Nesd fragments, and protein extracts were analyzed by Western blotting using an anti-myc (α-myc) antibody (input). The same extracts were then subject to PrA pull-down assay, and the presence of myc-tagged fragments in the pull-down was detected by Western blotting. The numbers on the left indicate the sizes in kilodaltons of the molecular mass markers.