Figure 5

Liver tissue caspase activation in untreated and ConA-treated C57Bl/6 mice. C57BL/6 mice were injected with (A) PBS or (B-D) 12 mg ConA/kg. Liver specimens were collected (B) 3, (C) 6, or (D) 12 hr post-injection and processed immediately for caspase activity. For each condition, liver specimens were incubated at 37°C in oxygenated KH buffer in the presence of 74 μM Ac-DEVD-AMC with (dashed line) and without (solid line) 43 μM zVAD-fmk for 30 min. The cleavage reaction was quenched with tissue disruption. The supernatants were separated via HPLC and analyzed for Ac-DEVD-AMC (R _t, ~3 min) and free AMC (R _t, ~18 min) peaks as described in the Materials and Methods. In Panel E, liver specimens were treated in vitro with 8 μM dactinomycin for 60 min prior to the addition of Ac-DEVD-AMC with (dashed line) and without (solid line) zVAD-fmk.