American Society of Hematology

Fig. 4.

$Caspase activation accompanies apoptosis but not platelet activation. Platelets were activated with calcium ionophore A23187 (2 μmol/L) for 15 minutes at 37°C. For comparison, control and apoptotic (6 hours post UVB-treatment) Jurkat cells were examined in parallel with platelets. Platelet activation and apoptosis were confirmed by monitoring cell shrinkage (decreased forward scatter) and PS externalization by FACS analysis as detailed in the text. Cell lysates were analyzed by SDS-PAGE and Western blotting (50 μg protein/lane) with antibodies against caspase-9 (A) and caspase-3 (B). Fifty micrograms of lysate were also assayed against LEHD-AFC and DEVD-AFC to detect caspase-9–like (A, bottom panel) and caspase-3–like activity (B, bottom panel). In (C), control and A2387-treated platelets were subjected to subcellular fractionation. The cytosolic (S15) and mitochondrial (P15) fractions (40 μg protein) were then subjected to SDS-PAGE and Western blotting with anti-cytochrome c antibodies. The presented data are representative of 3 independent experiments.$

Caspase activation accompanies apoptosis but not platelet activation. Platelets were activated with calcium ionophore A23187 (2 μmol/L) for 15 minutes at 37°C. For comparison, control and apoptotic (6 hours post UVB-treatment) Jurkat cells were examined in parallel with platelets. Platelet activation and apoptosis were confirmed by monitoring cell shrinkage (decreased forward scatter) and PS externalization by FACS analysis as detailed in the text. Cell lysates were analyzed by SDS-PAGE and Western blotting (50 μg protein/lane) with antibodies against caspase-9 (A) and caspase-3 (B). Fifty micrograms of lysate were also assayed against LEHD-AFC and DEVD-AFC to detect caspase-9–like (A, bottom panel) and caspase-3–like activity (B, bottom panel). In (C), control and A2387-treated platelets were subjected to subcellular fractionation. The cytosolic (S15) and mitochondrial (P15) fractions (40 μg protein) were then subjected to SDS-PAGE and Western blotting with anti-cytochrome c antibodies. The presented data are representative of 3 independent experiments.

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