FIG. 3.

Activation of caspase-3, caspase-8, and caspase-9 in MNV-1-infected RAW264.7 cells. (A) Equivalent amounts of protein from serial collection time points in mock (24 h p.i.)- and MNV-1-infected RAW264.7 cells were loaded onto a polyacrylamide gel and subjected to electrophoresis. Unprocessed caspase-8 (55 kDa) was detected by Western blot analysis throughout the experiment (lanes 1 to 7). A corresponding band representing cleaved caspase-8 (18 kDa) was not detected. Unprocessed caspase-3 and caspase-9 were detected at earlier time points and in mock-infected cells (lanes 1 to 4) at 31 and 46 kDa, respectively. Cleaved caspase-9 and caspase-3 were observed starting at 12 and 16 h p.i., respectively, in MNV-1-infected cells (lanes 4 to 7). MW, molecular weight (kDa). (B) Fluorometric analysis of caspase-3, caspase-8, and caspase-9 activities in MNV-1-infected RAW264.7 cells collected at serial time points. Caspase-3, caspase-8, and caspase-9 activities were determined by incubation of cell lysates with AFC-conjugated caspase-specific peptide substrates. Proteolytic activities were quantified by measuring fluorescence at 505 nm. After normalization for protein content, the final values of caspase activity were derived by subtracting background fluorescence from the corresponding mock-infected RAW cell lysates. Data represent the mean values obtained in two replicate experiments. RFU, relative fluorescence units.