Fig. 7. Influence of nuclear localization on ability of Ect2 N-terminal fragments to interfere with cytokinesis. (A) The myc-tagged Ect2 constructs, Ect2_1-420, Ect2_1-388, Ect2_1-370, Ect2_1-360, Ect2_1-333, Ect2_1-333 containing three SV40 NLS motifs (NLS 1-333) and Ect2_1-388 with a mutated NLS (NLS mut 1-388) were transfected for 48 hours into HeLa S3 cells before fixation with paraformaldehyde and permeabilization with Triton X-100. Cells were then stained with anti-myc 9E10 antibodies and DAPI and analyzed for the presence of either a single nucleus or binucleation (and occasionally multi-nucleation). Histogram shows the results of three independent experiments (300 cells each) and bars indicate s.d. The localizations of the various constructs in interphase cells are indicated as N (nuclear), N/C (nuclear and cytoplasmic) and C (cytoplasmic). (B) Equal amounts of cell lysates, prepared from mitotic cells transfected with the constructs described in A, were separated by SDS-PAGE and probed by western blotting with anti-myc 9E10 antibody. Anti--tubulin detection is shown as a loading control. (C) HeLa S3 cells were transfected for 36 hours with the indicated myc-Ect2 constructs. Cells were then fixed with paraformaldehyde and permeabilized with Triton X-100, before they were stained with anti-myc 9E10 antibodies (red, left), anti--tubulin antibodies (green). Merged images including DNA stained with DAPI (blue) are shown on the right. Bar, 10 µm.