Fig. 4.
FRAP analyses of mScarlet-Dsn1 during mitosis. (A) Immunoblot analysis of cells in which endogenous CENP-C was replaced with GFP-fused CENP-CWT (WT), CENP-CΔ648-676 (Δ648-676), or CENP-CT651A (T651A) (left three lanes). In each cell line shown in the right three lanes, mScarlet-fused Dsn1 was also replaced with endogenous Dsn1. The indicated antibodies were used for immunoblot analyses. (B) Localization of CENP-C-GFP (green) and mScarlet-Dsn1 (red) in cells. DNA was stained with DAPI (blue). The scale bar indicates 10 μm. The graph shows the Dsn1 intensities at the kinetochores in each cell line. (C) FRAP analysis of mScarlet-Dsn1 during mitosis. Left panels are images before bleaching (pre-bleach). The boxed area was bleached, and signal recovery is shown at the indicated time points. The black scale bar indicates 10 μm. (D) Solid lines display the means of quantitative FRAP measurements for mScarlet-Dsn1 from at least five cells for each line. The colored area between the two dashed lines indicates the standard deviation for each time point