The specificity is due to the use of a protein instead of a peptide substrate, and even though the VEID sequence is derived from the caspase-6 cleavage site in lamin A (aa227-230), we show that the Kcat/Km value for lamin A is more than 10fold higher than for VEID, indicating that lamin A is a better substrate for caspase-6. A similar effect has been shown for proteases involved in blood Figure 6 . Caspase-6 activity can be quantified by immunofluorescent staining for cleaved lamin A. A: C6wt and C6ko MEFs were stressed with camptothecin, stained with an antibody against cleaved lamin A and analyzed on an automated imaging platform. Nuclei were counterstained with DAPI, and identified by the software (blue circles). Debris (red circles) was not analyzed. The signal intensity in the cleaved lamin A channel was quantified in a ring around the nucleus (green circles). B: Quantitation of the perinuclear staining from cleaved lamin A is graphed. Untreated C6wt MEFs were normalized to 100% and the fold change in stressed C6wt, stressed and non-stressed C6ko MEFs are compared. Error bars are the SEM of N$3 of 3 independent experiments. Statistical significance was assessed by 2way ANOVA and post-hoc Bonferroni comparisons: *** p,0.0001. doi:10.1371/journal.pone.0027680.g006 coagulation [34, 35] , where substrates are bound outside the active site of the protease (exosite). There has been speculation about the presence of exosites in caspases [36] , but their existence has not been proven conclusively yet. The example of exploiting protein substrate specificity for the development of an activity assay could be used for the development of more specific assays for other members of the caspase family, such as p23 for caspase-7 [3] .