Fig. 4. Caspase activity is crucial for influenza A virus replication. MDCK cells were transfected with control vector or plasmids encoding the caspase inhibitory protein XIAP or procaspase 3. Transfection efficiency was determined upon parallel transfection of a GFP-expressing plasmid in FACS analysis to be 60%. Twenty-four hours post-transfection, cells were infected with influenza A virus (m.o.i. = 1). Supernatants were collected and cells were harvested 24 h p.i. (A) Proteins of cell lysates were separated by SDS–PAGE and blotted onto nitrocellulose. Membranes were incubated with an anti-PARP antibody to monitor caspase activity. Overexpression of GST–XIAP or procaspase 3 was verified by anti-GST or anti-caspase 3 antibodies (note that the caspase 3 antibody does not detect endogenous protein in MDCK cells). Equal loading of proteins was determined with an anti-ERK western blot. (B) Band intensity of cleaved PARP in (A) served as a marker for caspase activation levels. (C) Supernatants were assayed for virus content in common plaque titrations. Virus titres are shown relative to the DMSO control, which was arbitrarily set as 100%.