Caspase-6 Activity Assessment in Cell Culture Figure 3. Increased sensitivity and highly improved specificity in a lamin-based caspase-6 activity assay. A+B: Quantities of down to 10 nM active caspase-6 can be detected with the novel lamin A-based caspase-6 assay while maintaining a signal-noise ratio of .3. C: The peptide inhibitor VEID-CHO shows a similar IC50 value in both the VEID- and the lamin A- based caspase-6 assays. D: The lamin A-based caspase-6 assay is highly specific, with no signal generated from C6ko cells even after 6 h of staurosporine stress. Error bars are the SEM of N$3 of 3 independent experiments. Statistical significance was assessed by 2-way ANOVA and post-hoc Bonferroni comparisons: *** p,0.0001, ** p,0.001. doi:10.1371/journal.pone.0027680.g003 would not necessarily change upon inhibitor treatment, or immunoprecipitation of active caspases with biotinylated zVAD- fmk, which depends on efficient precipitation and Western blotting for quantification [23]. Our newly developed ELISA method, however, could not be directly applied to neuronal cultures, since lamin A is not expressed in embryonic mouse brain [31]. Neurons in early stages of development up until postnatal day 5 express lamins of the B1 and B2 subtypes, which differ from the lamin A and C sequence at the caspase cleavage site (VEVD in B-type lamins and VEID in lamins A and C (Fig. 4A) [24,31]). Furthermore, the cleavage of lamins B1 and B2 at this site is not specific for caspase-6, as has been shown in a caspase-6-deficient cell line [24]. In agreement with these findings, we observed cleavage of lamin B1 in primary Table 1. Km and kcat values for caspase-6 substrates. Km kcat kcat/Km VEID 30.962.2 mM lamin A 14.1162.592 nM 4.360.12 sec21 139 200 M21 sec21 0.05760.0025 sec21 4 055 043 M21 sec21 doi:10.1371/journal.pone.0027680.t001 cortical neurons derived from both C6wt and C6ko mice at embryonic day 16.5 (Fig. 4B), while lamins A and C were not detected (data not shown). To overcome this obstacle, we decided to spike C6wt and C6ko neuronal lysates with purified lamin A protein, and after incubation at 37uC to allow for its cleavage by endogenous caspase-6 activity, we subjected the samples to our ELISA assay. We detect a significant increase in cleavage of lamin A protein when the incubation was performed in the presence of extracts derived from camptothecin-stressed C6wt neurons, whereas no increase in cleaved lamin A signal was observed in the presence of camptothecin-stressed C6ko neuronal extracts (Fig. 4C). The ELISA method is therefore also suitable to detect caspase-6 activity in samples that do not contain endogenous lamin A or C protein. Detection of caspase-6 activity in the absence of endogenous lamin cleavage Caspase-6 activity can be localized to the nucleus, which is commonly associated with cell death and the cleavage of nuclear substrates such as lamin A, whereas a cytoplasmic localization of active caspase-6 as it is the case in neurodegeneration does not result in immediate apoptosis [5,21,32]. We therefore decided to test whether spiking of cell extracts with pure lamin A protein can detect active caspase-6 at early timepoints, before its translocation PLoS ONE | www.plosone.org 5 November 2011 | Volume 6 | Issue 11 | e27680