Chk2 phosphorylates INCENP-Ser91 in late midbodies. (A) Localization of phospho-Chk2-Thr383 (pChk2-Thr383) or phospho-Chk2-Thr68 (pChk2-Thr68) in BE cells. (B) Immunoprecipitation (IP) of Chk2 and INCENP in cytokinesis-enriched BE cell extracts. (C–E) Chk2 in vitro kinase assays. Autoradiography (32P, top) and Western blot analysis (WB) or Ponceau staining (bottom) of the same gel. Asterisks, predicted molecular weights. (F) Alignment of INCENP protein sequences. Human Ser91 is boxed and marked by asterisk. (G–O) Localization and mean intensity of phosphorylated INCENP-Ser91 (pINCENP-Ser91) or V5-INCENP in asynchronous BE cells, or after treatment with 10 µM Chk2 inhibitor II (inhII) in cytokinesis (L and M). Values represent mean ± SD from n cells. Values in control were set to 1. ***, P < 0.001 (Student’s t test). Tubulin values indicate midbody thickness. Insets show 1.6× magnification of the midbodies. S, serine. Scale bars, 5 µm.
