Chmp4c binds to ZW10. (A–C) GST-pull downs (A and C) and coimmunoprecipitation (coIP; B) from mitosis-enriched cells. ZW10, GST, or Chmp4c were detected by Western blotting. Ab, antibody; nocod, nocodazole. (D) Coomassie staining of GST proteins. (E) Western blot analysis (top) and ponceau staining (bottom) of GST pull-downs from mitosis-enriched cells. Amino acid numbers of Chmp4c fragments or mutations are shown in parentheses. (F) GST pull-downs with the use of purified proteins. 0.5 µg His-Chmp4c was incubated with 1 µg GST or GST-ZW10. His (top) or GST (bottom) was detected by Western blotting. His-Chmp4c input was 50%. (G) Coomassie-stained gel of GST pull-downs with the use of purified proteins. 1 µg His-Chmp4c was incubated with 2 µg GST or 1 µg GST-ZW10. His-Chmp4c input was 50% and GST or GST-ZW10 inputs were 100%. Ab, antibody; nocod, nocodazole.