Sti is required for proper localization of Rho1 at the CF. (A) Drosophila S2 cells were treated with dsRNAs directed against kanamycin (control) or sti for 72 h and then fixed and stained to detect Rho1, tubulin, and DNA (blue). (B) GST::Rho1 variants were preincubated with GTP and then mixed with Sti1,150–1,331, Sti1,437–1,854, or Rhotekin RBD, translated, and radiolabeled in vitro. The mixtures were then pulled down using glutathione beads, separated on SDS-PAGE gels, transferred onto nitrocellulose membrane, and exposed to x-ray films. The Ponceau S staining of protein loading is shown at the bottom. The numbers on the left indicate the sizes in kilodaltons of the molecular mass marker. (C) S2 cells stably expressing Myc alone or Myc-tagged Sti full length (StiFL), StiΔCNH, or a kinase-dead version of Sti (StiKD) were treated with dsRNAs directed against either kanamycin (kana) or the 3′ UTR of sti for 72 h. The number of multinucleate cells was then counted and plotted. Only Myc-positive cells were counted, and >600 cells were counted in each experiment. (D) Quantification of the cells showing aberrant Rho1 localization from experiments performed as described in C. At least 120 cells were counted in each experiment. (E) Cells expressing the transgenes described in C and depleted of endogenous Sti were stained to detect Rho1, Myc, and tubulin. Bars, 10 µm. n = 4; *, P < 0.05 (Mann–Whitney U test). Error bars indicate standard deviations.
