Caspase-6 Activity Assessment in Cell Culture Figure 2. VEID, but not lamin A+C, is cleaved in the absence of caspase-6. A: Caspase-6 protein (full-length, 32 kDa) is detected in MEFs generated from C6wt, but not C6ko mice. B: Endogenous lamin A protein (70 kDa) is cleaved in wt, but not C6ko MEFs after staurosporine stress for 4 h or longer. The antibody cross-reacts with full-length lamin C (60 kDa), and the cleaved band at 28 kDa has the same size for both lamin A+C (lower panel). C: MCF-7 cells express caspase-6, but not caspase-3 protein, whereas both C6wt and C6ko MEFs contain both caspases. hu: human, m: mouse, ns: non-specific band. D: MCF-7 cells were stressed with 5 mM camptothecin for different amounts of time and the cleavage of endogenous lamin A and C proteins was monitored by Western blotting with antibodies antibodies #2031 (full-length lamin A+C) and #2032 (C-terminal fragments). E: Schematic representation of lamin A and C and the caspase-6 cleavage site at AA 230. The N-terminal fragments generated by caspase- 6 cleavage (red) have the same size (28 kDa) for both lamin A+C. F: C6wt or C6ko MEFs were stressed with 50 nM staurosporine for different amounts of time, lysates were generated and analyzed for cleavage of VEID-Afc. C6wt cells show a significant increase in fluorescence at each timepoint, the fluorescence signal obtained from C6ko lysates only reach a statistically significant difference from baseline after 4 h. C6ko MEFs stressed with staurosporine for 4 h or more show the same levels of VEID proteolysis as wt cells. Error bars are the SEM of N = 3 of 4 independent experiments. Statistical significance was assessed by 2-way ANOVA and post-hoc Bonferroni comparisons: *** p,0.0001, ** p,0.001, * p,0.01. doi:10.1371/journal.pone.0027680.g002 endogenous lamin A protein is a highly specific readout for the quantification of caspase-6 activated during staurosporine-induced apoptosis. Kinetic measurements of either Ac-VEID-AFC or lamin A cleavage by fluorescence or our newly developed ELISA allowed comparison of the kinetic parameters of caspase-6 for the two substrates. We find that the full-length lamin A protein has a more than 1000fold lower Km than its cleavage site peptide VEID (Table 1). Although the kcat value is also decreased, the kcat/Km is still more than 10fold higher for lamin A (Table 1). This indicates that the binding between lamin A and caspase-6 is tighter, which might be mediated by domains outside the cleavage site. Such binding sites could also be responsible for the observed specificity of caspase-6 for the full-length protein substrate. The Km and kcat values for VEID are furthermore in good agreement with previously reported data [4]. Quantitation of caspase-6 activity in neuronal lysates Caspase-6 is postulated to be involved in neuronal degeneration and apoptosis [16,17,18,20,22,30], and detection of its activity in neuronal cultures is therefore of paramount interest. Previous studies have frequently used TUNEL assays as a measure of apoptosis, looked at VEIDase activity or the presence of the active caspase-6 fragment by Western blotting or immunostaining and assessed the presence of cleaved caspase-6 substrates [8,18,20,21,22]. The most specific methods to assess the effect of drug treatments on caspase-6 activity developed so far use antibodies against the active caspase-6 fragment, levels of which PLoS ONE | www.plosone.org 4 November 2011 | Volume 6 | Issue 11 | e27680