JCS03032 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Specificity of the anti-N-terminal Ect2 antibody. Equal amounts of U2OS whole-cell lysate were separated by SDS-PAGE and transferred onto nitrocellulose membranes. These membranes were probed with pre-immune serum, immune serum and affinity purified Ect2 antibodies, as indicated.

• Supplemental Figure 2 -

  Fig. S2. Specificity of the anti-C-terminal Ect2 antibody. HeLa S3 cells were treated with GL2 and Ect2 siRNAs for 48 hours. Subsequently, cell lysates were prepared and proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes. These were probed with anti-C-terminal-Ect2 antibodies (upper panel) and, as a loading control, with anti-α-tubulin antibodies (lower panel).

• Supplemental Figure 3 -

  Fig. S3. Citron kinase is essential for cytokinesis in HeLaS3 cells. (A) HeLa S3 cells were treated for 48 hours with control (GL2) or Citron kinase siRNAs and then fixed and permeabilized with paraformaldehyde and Triton X-100. Cells were stained with anti-α-tubulin antibodies (green) and DNA was stained with DAPI (blue). Bar, 10 μm. (B) Cells treated as in A were extracted with HEPES lysis buffer and proteins were separated by SDS-PAGE. They were then transferred to nitrocellulose membranes and probed with anti-Citron kinase antibodies, and with anti-α-tubulin antibodies to show equal loading.

• Movie 1 -

  Movie 1. Control HeLa S3 cells were treated with control (GL2) siRNA for 24 hours. Images were collected every 2 minutes and played back at 12.5 frames/second (1500× real time).

• Movie 2 -

  Movie 2. Ect2 siRNA. HeLa S3 cells were treated with Ect2 siRNA for 24 hours. Images were collected every 2 minutes and played back at 12.5 frames/second (1500× real time).

• Movie 3 -

  Movie 3. Myc-Ect2_1-333 overexpression. An inducible myc-Ect2_1-333 stable cell line was induced with tretracycline for 8 hours, before filming was started. Images were collected every 2 minutes and played back at 12.5 frames/second (1500× real time).