Figure 2

SDS-PAGE analysis of engineered caspase-3 proteins. A. Expression and Western blot analysis of engineered caspase-3 proteins. Cell lysates (20 μg) were prepared using an identical method, and loaded onto each well. M, molecular weight size marker; lane 1, uninduced E. coli lysate expressing wild-type caspase-3; lane 2, IPTG-induced E. coli lysate expressing wild-type caspase-3 (I); lane 3, IPTG-induced E. coli lysate expressing Δ28-caspase-3 (II); lane 4, IPTG-induced E. coli lysate expressing Δ28/175TS-caspase-3 (III); lane 5, IPTG-induced E. coli lysate expressing 28TS/175TS-caspase-3 (IV); lane 6, IPTG-induced E. coli lysate expressing 28TS/180TI-caspase-3 (V). The right and left images were obtained after coomassie blue staining and Western blot analysis, respectively. B. Treatment of caspase-3 precursors with wild-type caspase-3 (I). After each caspase-3 precursor was treated with wild-type caspase-3 (I) at RT for 18 h, 20 μl of each sample was heat-denatured and analyzed by SDS-PAGE. M, molecular weight size marker; lane 1, III; lane 2, III processed with I; lane 3, IV; lane 4, IV processed with I; lane 5, V; lane 6, V processed with I; lane 7, C163S caspase-3; lane 8, C163S caspase-3 treated with I. C. Activation of caspase-3 precursors by thrombin. Caspase-3 precursors were treated with thrombin at 4°C for 18 h, and 20 μl of each sample was heat-denatured and analyzed using PAGE. M, molecular weight size marker; lane 1, III; lane 2, III processed with thrombin (VI); lane 3, IV; lane 4, IV processed with thrombin (VII); lane 5, V; lane 6, V processed with thrombin (VIII); lane 7, C163S caspase-3; lane 8, C163S caspase-3 processed with thrombin.