Fig. 2

Caspase inhibitor zVAD-fmk is stable and prevents caspase activity. Medium alone (A) or neuronal cultures (B) were supplemented with N-benzyloxycarbonyl-Val-Alaaspartyl-fluoromethyl ketone (zVAD-fmk; 100 µM). After the indicated time points, supernatant was removed and incubated in different dilutions with recombinant caspase-3 (30 ng/ml). Then, caspase-3 activity was measured by Ac-Asp-Glu-Valaspartyl-aminotrifluoromethyl coumarin (DEVD-afc) cleavage. Data are means from five determinations. Error bars are smaller than the data symbols. (C) Enzymatic activities of recombinant caspase-2 and caspase-3 (0.6 µg/ml) were measured with the peptide substrates DEVD-afc and Ac-Val-Asp-Val-aspartylaminotrifluoromethyl coumarine (VDVAD-afc). Data are means ± standard deviation (SD) from three measurements. (D) AcAsp-Glu-Val-Asp-aldehyde (DEVD-CHO) and zVAD-fmk in concentration from 0.1 nM to 100 µM were incubated with recombinant caspase-2 (15 ng/ml) or caspase-3 (30 ng/ml). Then, enzymatic activity for caspase-2 (VDVAD-afc) and caspase-3 (DEVD-afc) was determined. Data are means from six determinations. Error bars are smaller than the data symbols. (E) Neurons were treated with colchicine (1 µM) in the presence or absence of zVAD-fmk (100 µM) or with zVAD-fmk alone. After the indicated time points, cells were lysed and lysates were incubated in vitro in the presence or absence of DEVD-CHO (50 nM) for 5 min at room temperature. Then, cleavage activity was measured with the substrates DEVD-afc and VDVAD-afc. Data are means ± SD from three experiments.