Sti localizes to the CF via interaction of its CC region with actin and myosin. (A) Schematic representation illustrating Sti protein domains and the localization of different Sti fragments tagged with GFP. (B) Drosophila S2 cells stably expressing various GFP-tagged Sti fragments (indicated at the top) were fixed and stained to detect GFP, tubulin, and DNA (blue). The arrow in the StiFL image marks a midbody remnant. In the CC1 image, the arrowhead marks the spindle midzone, whereas the arrow indicates CC1 localization to centrosome and astral microtubules. GFP::CC2a localization is shown in early and late telophase and after treatment for 72 h with a dsRNA directed against the CNH region (sti RNAi). Bars, 10 µm. (C) S2 cells stably expressing either the Sti CC2a fragment tagged with PtA or PtA alone were transfected with a Myc::Sqh construct for 48 h, and then protein extracts were used in a PtA pull-down assay. The extracts and pull-downs were analyzed by Western blotting to detect Myc::Sqh, actin, Rho1, and PtA. (D) S2 cells stably expressing PtA::StiCC2a were transfected with a Myc::Sqh construct for 48 h and, 5 h before collection, treated either with Cytochalasin D (Cyto D) or its solvent DMSO. Protein extracts were then subject to PtA pull-down assay, and extracts and pull-downs were analyzed by Western blotting to detect Myc::Sqh, actin, Rho1, and PtA. Numbers on the left indicate the sizes in kilodaltons of the molecular mass markers.