Fig. 5.
Borealin-microtubule interaction contributes to robust phosphorylation of the kinetochore substrates by the CPC. a Schematic of Borealin siRNA mediated knockdown rescue experiment. HeLa-TReX cells stably expressing LAP-BorealinWT or MTBM were treated with Borealin 3’UTR siRNA and cells were immunostained in first mitosis after knockdown of endogenous Borealin. Kinetochore phosphorylation was assessed by immunostaining with b DSN1 pS109, d Knl1 pS60, h Hec1 pS69, antibodies. Green color in merge images indicates LAP-Borealin localization. f Chromatin phosphorylation was assessed by immunostaining with histone H3 pS10. Box and whisker graphs of normalized intensity from c DSN1 pS109 (n = 103 (from 8 cells) for WT and n = 73 (from 9 cells) for MTBM), e Knl1 pS60 (n = 61 for WT and n = 72 for MTBM, from eight cells per condition), g H3 pS10 (n=18 for WT and n=11 for MTBM) and i Hec1 pS69 (n = 68 (from 7 cells) for WT and n = 73 (from 6 cells) for MTBM, from at least six cells per condition), staining. All representative immunofluorescence images and quantitation data are from 1 of 2 independent repeats. Statistical analysis was performed using Mann Whitney Test, ***P < 0.0001 and ns P > 0.05. All Box and whisker plots represent the median (central line), 25th–75th percentile (bounds of the box) and 5th–95th percentile (whiskers). Scale bar is 5 μm