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. 1998 Oct 5;143(1):225–239. doi: 10.1083/jcb.143.1.225

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A fraction rich in endogenous caspases fails to induce apoptotic events in added nuclei. (A) Protocol used to prepare fractions 1–3 by heparin–agarose chromatography of roller S/M extract. (B) Active caspases were labeled with zEK(biotin)D-aomk in roller S/M extract and fraction 2, but could not be detected in Fr-3. The bands in the upper portion of the gel are cellular proteins that bind to the streptavidin probe. (C) PARP and lamin A are efficiently cleaved by roller S/M extract and by Fr-2. Fr-3 has low levels of PARP cleavage activity that are abolished by pretreatment with DEVD-fmk. (D) Pulsed field gel electrophoresis. High molecular weight DNA fragments were produced in HeLa nuclei incubated in roller S/M extracts and Fr-3 (caspase-deficient), but not in nuclei incubated in buffer or Fr-2, which contains high levels of active caspases. Lane M, DNA markers (sizes shown in kilobase pairs). (E) Conventional agarose gel electrophoresis. An oligonucleosomal DNA ladder is formed in nuclei incubated in roller S/M extracts and Fr-3, but not in nuclei incubated in buffer or Fr-2. Lane M, DNA markers (sizes shown in base pairs). (F) Fr-3 induces very strong morphological apoptosis even when all detectable caspase activity is abolished by pretreatment with DEVD- fmk. In contrast, Fr-2, which has a distribution and concentration of caspases essentially identical to S/M extract, does not induce morphological apoptosis in HeLa nuclei.