Caspase activation following CD40 activation of mature DCs. (A) Mature DCs were activated for 4 hours with 5 μg/mL anti-CD40 (B-B20) or isotype control before cell lysis. Western blot was performed on protein extracts with anti-caspase-8, anti-caspase-9, and anti-caspase-3. Furthermore, caspase-8 activation was analyzed at various times. For cytochrome c release experiments, cytosolic and mitochondrial extracts were prepared (see “Materials and methods”) from mature DCs activated as described. Western blot was performed on protein extracts with anti-cytochrome c antibody. Similar results were obtained in 2 other independent experiments. (B) DCs were activated for the indicated times with agonistic anti-CD40 (B-B20) or isotype control. A fluorescent caspase inhibitor peptide was added 1 hour before flow cytometry. These results are the mean ± SEM of 3 independent experiments. (C) DCs were preincubated for 2 hours with Z-LEHD-FMK (caspase-9 inhibitor) or Z-IETD-FMK (caspase-8 inhibitor), then treated for 6 hours with anti-CD40 (B-B20) or isotype control before analyzing apoptosis as described. Results are the percentage inhibition of anti-CD40-induced apoptosis as compared to the isotype control. These results are the mean ± SEM of 3 independent experiments.