American Society of Hematology

Figure 1

$Figure 1. NPM inhibits and interacts with caspase-6 and -8. (A) Western blot analysis for cleaved caspases in S-100 cytosolic fractions extracted from HEK293T cells and incubated with cytochrome c (300nM) and/or deoxyadenosine triphosphate (dATP; 900nM), in the presence or absence of recombinant NPM (5μM) for 3 hours at 30°C. (B) S-100 cytosolic fractions from HEK293T were incubated with 1 unit of the various indicated recombinant active caspases (or 32 units for recombinant active caspase-8) in the presence or absence of 5μM recombinant His-tagged NPM for 1 hour at 30°C. Caspase activity was measured colorimetrically using pNA-taggd synthetic caspase substrates. (C) Western blot analysis of caspase-8 activity. Purified active recombinant caspase-8 (5nM) was incubated with 5nM to 5μM recombinant NPM and 150nM substrate recombinant procaspase-3 for 3 hours at 30°C. (D) Western blot analysis for caspase-6 activity. Purified recombinant caspase-6 (30nM) was incubated with 30nM to 3μM recombinant NPM and 625nM substrate GST-tagged lamin A for 3 hours at 30°C. (E) GST pull-down assay using equivalent amounts of GST or GST-NPM for cleaved caspase-6 and -8 in cytochrome c–treated HEK293T cytosolic extracts. (F) MN9D cells were treated with 500μM MPP for 12 hours, after which the cells were harvested and the total cell lysates extracted. Immunoprecipitation was carried out using antibodies against NPM, caspase-6, caspase-8, or hemagglutinin (HA; control). (G) Top: Western blot analysis for NPM in cytosolic or nuclear fractions of HeLa cells exposed to 0.40μM actinomycin D for 0 to 16 hours. Bottom: Cytosolic fractions collected were normalized to the same protein concentration and subjected to immunoprecipitation using antibodies against NPM or HA as a control. (H) Whole-cell lysate from apoptotically induced HEK293 cells was used in GST pull-down assay using either empty GST beads, GST-tagged full-length NPM, or its various deletion mutants. Immunoprecipitates were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and subjected to immunoblotting using anti-NPM, anti–caspase-6, or anti–caspase-8 antibody. (I) Domain structure of the full-length GST-NPM protein and the various deletion mutants used in the GST pull-down in panel H. Hed indicates heterodimerization domain; HoD, homodimerization domain; and NoLS, nucleolar localization signal. Arrows indicate the exact boundary of deletion in the various mutants.$

NPM inhibits and interacts with caspase-6 and -8. (A) Western blot analysis for cleaved caspases in S-100 cytosolic fractions extracted from HEK293T cells and incubated with cytochrome c (300nM) and/or deoxyadenosine triphosphate (dATP; 900nM), in the presence or absence of recombinant NPM (5μM) for 3 hours at 30°C. (B) S-100 cytosolic fractions from HEK293T were incubated with 1 unit of the various indicated recombinant active caspases (or 32 units for recombinant active caspase-8) in the presence or absence of 5μM recombinant His-tagged NPM for 1 hour at 30°C. Caspase activity was measured colorimetrically using pNA-taggd synthetic caspase substrates. (C) Western blot analysis of caspase-8 activity. Purified active recombinant caspase-8 (5nM) was incubated with 5nM to 5μM recombinant NPM and 150nM substrate recombinant procaspase-3 for 3 hours at 30°C. (D) Western blot analysis for caspase-6 activity. Purified recombinant caspase-6 (30nM) was incubated with 30nM to 3μM recombinant NPM and 625nM substrate GST-tagged lamin A for 3 hours at 30°C. (E) GST pull-down assay using equivalent amounts of GST or GST-NPM for cleaved caspase-6 and -8 in cytochrome c–treated HEK293T cytosolic extracts. (F) MN9D cells were treated with 500μM MPP for 12 hours, after which the cells were harvested and the total cell lysates extracted. Immunoprecipitation was carried out using antibodies against NPM, caspase-6, caspase-8, or hemagglutinin (HA; control). (G) Top: Western blot analysis for NPM in cytosolic or nuclear fractions of HeLa cells exposed to 0.40μM actinomycin D for 0 to 16 hours. Bottom: Cytosolic fractions collected were normalized to the same protein concentration and subjected to immunoprecipitation using antibodies against NPM or HA as a control. (H) Whole-cell lysate from apoptotically induced HEK293 cells was used in GST pull-down assay using either empty GST beads, GST-tagged full-length NPM, or its various deletion mutants. Immunoprecipitates were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and subjected to immunoblotting using anti-NPM, anti–caspase-6, or anti–caspase-8 antibody. (I) Domain structure of the full-length GST-NPM protein and the various deletion mutants used in the GST pull-down in panel H. Hed indicates heterodimerization domain; HoD, homodimerization domain; and NoLS, nucleolar localization signal. Arrows indicate the exact boundary of deletion in the various mutants.

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