Figure 6.

Efficient removal of checkpoint proteins from KTs requires dynein/dynactin. (A) HeLa S3 cells were treated as in Fig. 5 D but stained with anti-MAD2 antibody instead of anti-hSpindly antibody. (B) The percentage of metaphase cells treated as in A with MAD2-positive KT(s) was determined. Bar graph showing the results of three independent experiments (>30 cells each), with error bars indicating SD. (C) To perform an ATP reduction assay, cells were rinsed once with isotonic salt solution and then incubated with either isotonic salt solution with glucose or with sodium azide (Az) and 2-deoxyglucose (DOG). Cells were stained with anti-MAD2 antibody and CREST serum (top), anti-ZW10 and anti-Hec1 antibodies (middle), and anti-hSpindly and anti-Hec1 antibodies (bottom). All cells were simultaneously stained also with anti–α-tubulin antibody and DAPI (blue). (D) Cells were treated as in C, except that 3.3 µM nocodazole was added to the isotonic salt solution. Bars, 10 µm.