To this end, we tested lysates derived from staurosporine-stressed C6wt and C6ko MEFs with our newly developed ELISA and found a linear increase in signal over time in wt samples, whereas the signal from C6ko samples remained stable at background levels that were similar to the background seen in untreated C6wt (Fig. 3D ). This indicates that the amount of intracellularly cleaved, Figure 1 . The lamin A protein is a more specific substrate than its VEID peptide. A: 100 mM VEID-Afc was incubated with different amounts of caspase -3, -6 or -7 for 1 h at 37uC. Fluorescence generated by cleavage was monitored over time, and the initial, linear portion of the curve was used to calculate the reaction velocity. VEID is preferentially cleaved by caspase-6, but also cross-reacts with caspases -3 and -7 at higher concentrations. Error bars are the SEM of N$3 of 3 independent experiments. B: 5 mM VEID-Afc was incubated with 0.5 mM caspase-6 or different amounts of caspase-3 for 1 h at 37uC. The reaction velocity was calculated as in A. Even at this low concentration of VEID-Afc, the peptide substrate can be cleaved by caspase-3, and an 8 fold higher molar concentration of caspase-3 than caspase-6 results in a higher signal for VEID cleavage by caspase-3 than caspase-6. Error bars are the SEM of N = 3 independent experiments, statistical significance was assessed by 1-way ANOVA and posthoc Dunnett comparisons: *** p,0.0001. C: Pure lamin A protein was incubated with different amounts of caspase -3, -6 or -7 for 30 min at 37uC. Samples were separated by SDS-PAGE and both fragments of cleaved lamin A was detected by Western blotting with antibodies #2031 (full-length lamin A and N-terminal fragment) and #2032 (C-terminal fragment). No cleavage was observed with caspases -3 or -7, while caspase-6 generated lamin A fragments in a dose-dependent manner. A representative image of 3 independent experiments is shown. doi:10.1371/journal.pone.0027680.g001 endogenous lamin A protein is a highly specific readout for the quantification of caspase-6 activated during staurosporine-induced apoptosis.