Fig. 3. The MKlp1-MgcRacGAP and MKlp1–Aurora-B complexes are both required for localizing Ect2 to the central spindle. (A) HeLa S3 cells were treated with control (GL2), MKlp1, MgcRacGAP, Aurora-B and MKlp2 siRNAs for 36 hours before fixation and permeabilization with paraformaldehyde and Triton X-100, respectively. Subsequently, cells were stained with anti-Ect2 (left) and anti--tubulin (middle) antibodies. Merged images are shown on the right with Ect2 in red, -tubulin in green and DNA stained with DAPI in blue. Bars, 10 µm. In all cells, the focal plane was adjusted to visualize central spindle rather than cortex staining. (B) Cell lysates from anaphase/telophase synchronized HeLa S3 cells were used for immunoprecipitations (IP's) with anti-Ect2 antibodies (anti-Ect2) or pre-immune antibodies (IgG). Immunoprecipitates and input lysates (Extract) were then probed by western blotting with antibodies against the indicated proteins. (C) Immunoprecipitation using anti-MKlp1 antibodies. (D) Immunoprecipitation using anti-MgcRacGAP antibodies.