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Fig. 5. Inhibition of Bub1 and aurora kinase activity does not have a synergistic effect on kinetochore localization of BubR1. DLD-1 cells were transfected with control or Bub1 siRNA duplexes. 48 hours after transfection, the cells were exposed to nocodazole plus or minus ZM447439 for 1 hour then fixed and stained to detect Bub1, BubR1 or Mad2, centromeres/kinetochores (ACA), and the DNA. In one sample, the anti-BubR1 or anti-Mad2 antibody was omitted to define the background signal. Image stacks of mitotic cells were acquired, deconvolved, then projected and the fluorescence pixel intensities at individual kinetochore pairs measured. At least 60 pairs in three or more cells were quantitated. The Bub1/ACA (A), BubR1/ACA (B) and Mad2/ACA (C) ratios were then calculated and the value for each kinetochore pair plotted on a log scale. Dark grey, control; red, Bub1-RNAi alone; green, ZM447439 alone; yellow, Bub1-RNAi plus ZM447439; light grey, control without BubR1 or Mad2 primary antibody. P values were determined using a nonparametric ANOVA (Kruskal-Wallis) test followed by a Dunn's post-test; ns, not significant (P>0.05); *significant (P<0.05); **very significant (P<0.01). See Table 1 for means and s.e.m.
