FIG. 4.

(A) Caspase 3, but not caspase 6, cleaves partially purified ATM in vitro. Partially purified ATM pool (ATM-Q; 50 μg of total protein) was incubated with no caspase (−) or increasing amounts (33, 100, or 300 ng) of caspase 3 or caspase 6 for 30 min at 37°C. The cleavage of PARP by caspase 3 is shown in the left, lower panel, and the cleavage of lamin B by caspase 6 is shown in the right, lower panel. (B) Caspase 3-mediated cleavage of ATM in vitro yields the same proteolytic degradation pattern as that seen in HL60 cells undergoing apoptosis. Lane 1, 50 μg of nuclear extract from untreated HL60 cells; lane 2, 25 μg of nuclear extract from HL60 cells treated with 68 μM etoposide for 5 h; lane 3, 20 μg of partially purified ATM (ATM-Q) incubated with 300 ng of caspase 3; lane 4, 20 μg of untreated partially purified ATM (ATM-Q). Western blot analysis was performed with anti-ATM antiserum ATM-B. (C) The caspase 3-deficient cell line MCF-7 does not cleave ATM after treatment with TNF-α (30 ng/ml) and cycloheximide (CHX) (10 μg/ml) for 20 h. Cleavage of PARP in the apoptotic MCF-7 cells is shown in the right-hand panel.