Caspase-6 Activity Assessment in Cell Culture 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 post- hoc 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 luminescence [29]. To determine whether cleavage of lamin A is more sensitive than cleavage of the VEID peptide substrate in detecting low levels of active caspase-6, we incubated different concentrations of the enzyme in parallel either with Ac-VEID- AFC or purified lamin A protein and determined the amount of cleavage after 30 min by measuring the fluorescence in the sample (for Ac-VEID-AFC) or by subjecting the sample to an ELISA using the lamin A neo-epitope antibody (for lamin A cleavage). Comparison of the results showed that both assays perform at least equally well in detecting active caspase-6 concentrations down to 10 nM, with the lamin cleavage assay showing a linear concentration-response relationship down to the lowest caspase concentrations tested (Fig. 3A). Signal-to-noise ratios were at or above 3 for the ELISA assay for as low as 10 nM caspase-6, whereas the signal-to-noise dropped below 3 for the VEID-based assay at this concentration (Fig. 3B). Using a peptide inhibitor for caspase-6, VEID-CHO, both assays furthermore arrived at a similar IC50 value (lamin-based ELISA: 64620 nM, VEID-based assay: 5666 nM), indicating that the ELISA method is suitable to assess the inhibition of caspase-6 by small molecules or peptides. Overall, our method shows a slight increase in sensitivity over commonly used VEID cleavage methods and is able to reliably detect caspase-6 concentrations down to 10 nM. Next, we wanted to assess whether the lamin cleavage assay shows higher specificity than the VEID-based system for caspase-6 over other proteases that are activated after induction of apoptosis. 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, PLoS ONE | www.plosone.org 3 November 2011 | Volume 6 | Issue 11 | e27680