ATM kinase activity promotes caspase-8 activation. (A) Fas receptor levels were detected by flow cytometry analysis. Cells were incubated with anti-Fas antibodies followed by PE-conjugated secondary antibodies (dark lines). For each cell line, an incubation with PE-conjugated alone served as negative controls (light lines). (B) Caspase-8 expression was revealed by immunoblotting on extracts obtained by the indicated cell lines. Protein extract (80-100 μg) was separated by SDS-PAGE and transferred on nitrocellulose, and caspase-8 expression was revealed using specific antibodies. (C) Protein extracts from the indicated cell lines stimulated to undergo apoptosis with anti-Fas antibodies have been separated by SDS-PAGE, and caspase-8 was revealed by immunoblotting with specific antibodies. The arrows point to the entire protein, p55, as well as to the processing products p43 and p18. (D) Caspase-8 activity from the same extracts was measured by the hydrolysis of the caspase-8 substrate Ac-IETD-AMC.