BioMed Research International / 2009 / Article / Fig 2

Research Article

Activation of Cyclin-Dependent Kinase 5 Is a Consequence of Cell Death

Figure 2

Inhibition of calpain does not protect fibroblast C8 cells from CPT-induced cell death or activation of Cdk5. (a) C8 cells were exposed to 50  M CPT in the presence or absence of 15  M calpain inhibitor, PD, for 8 hours or 18 hours. The amount of cell death was measured by trypan blue exclusion. The error bars represent the standard deviation from at least three individual experiments. CPT killed 40% of the cells within 8 hours and 90% by 18 hours, but PD did not affect the toxicity. (b) Cells treated with 50  M CPT or CPT plus 15  M PD were collected and lysed by RIPA buffer. (A) Western blot of Cdk5 in equal amounts of cell lysates from cells treated with CPT or CPT PD for 18 hours showed an unchanged level of Cdk5 protein (32 kDa) during cell death. ((B) and (C)) Histone H1 kinase activity of Cdk5 immunoprecipitates was measured using histone H1 as substrate. The histone H1 phosphorylated by Cdk5 was detected by western blot (31 kDa). The kinase activity reflected the amount of cell death induced, following 8-hour (B) or 18-hour (C) treatment with CPT in the presence or absence of PD. PD did not affect the amount of histone kinase activity seen at 8 hours, though by 18 hours more was lost in the presence of PD than in its absence.

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