We therefore decided to investigate whether lamin A is a specific substrate for caspase-6. In particular, we were interested in the ability of other executioner caspases to cleave lamin A, since these are likely to show high activity in apoptotic extracts where accurate quantification of caspase-6 activity will be of interest. We first subjected the Ac-VEID-AFC substrate to digestion by caspases -3, -6 and -7, respectively. The amount of active enzyme used in each reaction was normalized to the concentration of active sites in the sample as determined by titration against the irreversible inhibitor zVAD-fmk (Fig. S1 ). As expected, the VEID peptide substrate, although it is best processed by caspase-6, still shows significant cleavage by caspase-3 and -7 (Fig. 1A ) at high substrate concentrations (100 mM). Lower substrate concentrations can be used to achieve greater specificity, however, the difference in kcat/Km for VEID is less than 3 fold between caspase -3 and -6 [4] . This results in a small concentration window that can be exploited to achieve selectivity for caspase-6, if the same amounts of active caspase-3 and -6 are present. However, this window will be lost if the sample contains a higher concentration of active caspase-3 than caspase-6. As shown in Figure 1B , an 8 fold molar excess of caspase-3 over caspase-6 is enough to lead to a significantly higher signal from the non-specific caspase-3, making VEID-based assays problematic for the use in apoptotic samples or other cell and tissue lysates with high levels of active caspase-3. Lamin A, on the other hand, only showed proteolytic processing when incubated with recombinant active caspase-6, not with the corresponding amounts of caspases -3 and -7 at concentrations up to 300 mM (Fig. 1C) . We therefore focussed on lamin A as a substrate for the development of a specific caspase-6 activity assay.