Basal ATM kinase activity regulates FLIP protein levels. (A) FLIP expression was revealed by immunoblotting on extracts obtained from the indicated cell lines. Protein extract (80-100 μg) was separated by SDS-PAGE and transferred on nitrocellulose, and FLIP expression was revealed using specific antibodies. The arrows point to FLIP-L and FLIP-S isoforms. (B) ATM-deficient L6 cells were stably transfected with shFLIP or with a scrambled oligo as control. Protein extracts from the indicated cell lines were probed for FLIP expression by immunoblotting as described in panel A. The arrows point to endogenous FLIP-L and FLIP-S. (C) The indicated cell lines were stimulated to undergo apoptosis with 250 ng/mL anti-Fas mAb. Apoptosis was determined by the analysis of DNA fragmentation upon PI nuclear staining 24 hours after anti-Fas treatment.