Expression of Ser91A V5-INCENP accelerates cleavage of the intercellular canal in cytokinesis. (A and B) Localization of Mklp1 and mean intensity at the midbody center in BE cells in late midbodies. (C–E) Localization of phosphorylated Aurora B-Ser331 (pAurora B-Ser331) and mean intensity at the midbody center in BE cells. (F, M, and Q) Western blot analysis of total INCENP, V5, Chk2 and actin in BE cells expressing GFP:INCENP(FB) or V5-INCENP. (G and H) Specificity of the anti-phospho-Chk2-Thr383 (pChk2-Thr383) and phospho-Chk2-Thr68 (pChk2-Thr68) antibodies. (I) Localization of phosphorylated INCENP-Ser91 (pINCENP-Ser91). BE cells were treated with 10 µM etoposide for 4 h. Damaged DNA is evidenced by γ-H2AX-staining. (J and K) Specificity of the anti-phospho-Ser91 antiserum. Where indicated, the anti-pINCENP-Ser91 antiserum (Ab) was incubated with the phosphorylated Ser91 peptide (phospho-Ser91, pSer91) or with the unphosphorylated (Ser91) synthetic peptide. Tubulin values indicate midbody thickness. Insets show 1.6× magnification of the midbodies. (L) Mean pINCENP-Ser91 intensity at the midbody center in BE cells. Values in control were set to 1. (N and O) Phase-contrast live-cell microscopy analysis of BE cells expressing WT or Ser91A V5-INCENP. Intercellular canals are shown by solid arrows. Time is from formation of an intercellular canal to canal cleavage (dotted arrows). Values represent mean ± SD from n cells. (P) Frequency of bi/multinucleate or prometaphase BE cells. Values represent mean ± SD from three independent experiments (n > 90). ***, P < 0.001 (ANOVA and Student’s t test). S, serine. Scale bars, 5 µm.