Fig. 3. (A) Mad2-GFP as a speckle in nda3-KM311 mutant. The cells were collected every hour during the synchronization at the restrictive temperature. After incubation for 6 hours (indicated by the arrowhead), the cells were released into the permissive temperature. Note that the samples were collected every 3 minutes after the point indicated by the arrowhead. Mad2-GFP was visualized under the microscope, and the percentage of the cells showing Mad2 as speckles versus total cell number was calculated. Over 100 cells were examined at each time point. (B) Mad2 is released from kinetochores after metaphase. A block and release experiment using the nda3-KM311 mutant was performed to stain tubulin by indirect immunofluorescence (red) and DNA by DAPI (blue). Mad2-GFP (green) was visualized in the same cells. (C) Slp1 and Mad2 protein expression after metaphase arrest. Slp1 and Mad2 protein expression in the nda3-KM311 mutant cells was analyzed 0, 3, 6, 9, 12 and 15 minutes after the release. The proteins were blotted using anti-Slp1 or anti-Mad2 antibody.