Fig. 7. Localization of Ndc80p and Spc24p in ndc10-1 cells. (A) Ndc80p and Spc24p are not associated with the kinetochore in ndc10-1 cells incubated at the restrictive temperature. Cells of ndc10-1 NDC80-6HA (lanes 1, 2, 7 and 8), ndc10-1 spc24-3HA (lanes 3, 4, 9 and 10) and ndc10-1 (control; lanes 5, 6, 11 and 12) grown at 23°C (lanes 1–6) were shifted to 37°C for 4 h (lanes 7–12). CEN3 association of Ndc80p-6HA (lanes 2 and 8) and Spc24p-3HA (lanes 4 and 10) was investigated by ChIP. ndc10-1 cells were employed as a control for the specificity of the immunoprecipitation (lanes 6 and 12) as well as binding to regions left (ChIII-L) and right (ChIII-R) of CEN3 DNA. Yeast lysates were also tested without precipitation (Load: lanes 1, 3, 5, 7, 9 and 11). (B) Cells of ndc10-1 NDC80-9Myc and ndc10-1 NDC80 were shifted from 23 to 37°C for 4 h. Fixed cells were analysed by indirect immunofluorescence using anti-tubulin and anti-Myc antibodies. DNA was stained with DAPI. The bottom panel shows the merged signals of the DNA (blue), tubulin (green) and Ndc80p-9Myc (red) stainings. Bar: 5 µm.