Clustering or kinetochore targeting of the CPC is insufficient to support the SAC. (A) Diagram of LAP-tagged hINCENP constructs. (B) Western blot of anti-GFP immunoprecipitation (IP) of the indicated constructs from mitotic HeLa cell extracts. (C) DoM for cells in taxol. Representative of n = 2 independent experiments. (D) Immunofluorescence of various INCENP constructs. Each construct was expressed over endogenous INCENP. For each construct, the left three panels are single-cell images; the right three are zoom-ins of a representative kinetochore pair indicated by white boxes. Bars: (single cell) 2 µm; (centromere/kinetochore) 0.25 µm. (E) DoM for cells in taxol. The maximum height for the density estimate of hINCENP ΔCEN was scaled independently. Representative of n = 2 independent experiments. (F) Each construct was expressed over endogenous INCENP and synchronized at the G2/M transition by treatment with the Cdk1-inhibitor RO-3306. After washout of the inhibitor, cells synchronously entered mitosis. Plots show the cumulative minimum DoM for each construct. Blue bars, arrested for at least 2 h; black bars, arrested for at least 10 h; n ≥ 90 cells per sample.