Fig. 4. Ect2 displacement from the central spindle does not interfere with cytokinesis. (A) HeLa S3 cells were grown on coverslips and transiently transfected for 36 hours with the indicated myc-Ect2 constructs before fixation and permeabilization with formaldehyde and Triton X-100. Cells were then stained with anti-myc 9E10 antibodies (middle) and antibodies recognizing a C-terminally located epitope on endogenous Ect2 (left). Merged images show transfected myc-Ect2 (9E10) in green, endogenous Ect2 in red and DNA stained with DAPI in blue. A control, non-transfected, cell is shown in the bottom row. Note that this antibody required a simultaneous fixation-permeabilization protocol, so that cortex localization of Ect2 was difficult to see. Bar, 10 µm. (B) Transiently overexpressed myc-Ect2 fragments were immunoprecipitated from HeLa S3 cells synchronized at anaphase/telophase, using the anti-myc 9E10 antibody (IP's). For control, an unrelated myc-tagged hWW45 construct was transfected and analyzed in parallel. Samples were then probed by western blotting with antibodies against the indicated proteins.