recombinant active caspase-9 Search Results


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  • 99
    Thermo Fisher anti caspase 9 antibody
    Phosphorylation of murine <t>caspase-9</t> CK2 decreases its susceptibility to cleavage by active caspase-8. A , purified caspase-9-FLAG was incubated with [γ- 32 P]ATP in kinase buffer in the presence or absence of purified recombinant CK2. Aliquots of the kinase reactions above were used as the source of substrate in cleavage assays using active, recombinant caspase-8 protein. Attenuated reactions were SDS-PAGE-resolved, transferred to nitrocellulose, and autoradiographed to visualize phosphorylated caspase-9 ( top panel ). The same membrane was then immunoblotted with an antibody against caspase-9 (Stressgen) to visualize total full-length and cleaved protein and determine relative cleavage by caspase-8 of unphosphorylated and phosphorylated substrates. B, in vitro translated [ 35 S]methionine-labeled mC-9 was used as substrate for phosphorylation by recombinant CK2. Kinase reactions were performed in the presence of cold ATP with or without CK2, following which caspase-8 cleavage reactions were carried out on aliquots of the kinase reactions. The results were visualized by autoradiography following resolution by SDS-PAGE. C , alternatively the kinase reactions were diluted in rehydration buffer and resolved by isoelectric focusing ( IEF , pH range 5–8) in the first dimension, followed by electrophoresis in the second dimension using an 8–16% gradient.
    Anti Caspase 9 Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore human recombinant caspase 9
    Exogenous cytochrome c /dATP fails to promote caspase-3 processing within platelet lysates. Cytosolic extracts were treated with cytochrome c and dATP or buffer alone and incubated at 37°C. (a) Western blot analysis of platelet lysates revealed a failure of cytochrome c /dATP to promote the processing of caspase-3. (b) By contrast, Jurkat T cell lysates showed progressive processing of the enzyme to the proteolytically active p17/p12 fragments. (c) On addition of human recombinant <t>caspase-9</t> and cytochrome c /dATP to fresh platelet lysates, derived from the same preparation used above, caspase-3 was now processed to its active p17/p12 subunits. Each Western blot had 50 μg of protein loaded per lane and was repeated twice.
    Human Recombinant Caspase 9, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore recombinant active caspase 9
    Caspase inhibition via cysteinyl oxidation in FPI affects caspase-3 preferentially to <t>caspase-9.</t> Cortical lysates were harvested at 24 h from FPI injured animals (2.4 atm) and assayed for caspase-3 and caspase-9 activity using Ac-DEVD-AMC and Ac-LEHD-AFC, respectively ( n = 6 animals). A , Effects of incubation with DTT, activated caspase-9 (3 U), or z-VAD-fmk on caspase-3 activity in cortex ipsilateral (right) and contralateral (left) to the FPI. DTT-treated samples exhibited significantly higher levels of caspase-3 proteolytic activity in the ipsilateral cortex compared with the contralateral cortex. B , Effects of incubation with DTT or z-VAD-fmk on caspase-9 activity in cortex ipsilateral (right) and contralateral (left) to the FPI. No significant differences in caspase-9 activity between the ipsilateral and contralateral cortex were observed with any treatment assayed.
    Recombinant Active Caspase 9, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Biomol GmbH recombinant active caspase 9
    In vitro inhibition of <t>caspase-9</t> activity by AMVIAP. Human caspase-9 activity was assayed in vitro with Ac-LEHD-AMC as a substrate. Caspase-9 was mixed with either GST at 2 μM (○), XIAP at 500 nM (+), and AMVIAP at 500 nM (•) or 5,000 nM (5 μM) (▴). Activity was estimated by LEHD-AMC cleavage. Each data series represents fluorescence emission (490 nm) at 1-min intervals for 120 min.
    Recombinant Active Caspase 9, supplied by Biomol GmbH, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    BioVision active recombinant human caspase 9
    In vitro inhibition of <t>caspase-9</t> activity by AMVIAP. Human caspase-9 activity was assayed in vitro with Ac-LEHD-AMC as a substrate. Caspase-9 was mixed with either GST at 2 μM (○), XIAP at 500 nM (+), and AMVIAP at 500 nM (•) or 5,000 nM (5 μM) (▴). Activity was estimated by LEHD-AMC cleavage. Each data series represents fluorescence emission (490 nm) at 1-min intervals for 120 min.
    Active Recombinant Human Caspase 9, supplied by BioVision, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Enzo Biochem recombinant caspase 9
    Inhibition assay of wild-type and designed XIAPs on <t>caspase-9</t> proteolytic activity by the Caspase-Glo® 9 Assay kit from Promega. The percent inhibitions are converted from the relative light units at different concentration of XIAP proteins. Lines connect data points to guide the eye.
    Recombinant Caspase 9, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 95/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Millipore recombinant caspase 9
    Caspase inhibition via cysteinyl oxidation in FPI affects caspase-3 preferentially to <t>caspase-9.</t> Cortical lysates were harvested at 24 h from FPI injured animals (2.4 atm) and assayed for caspase-3 and caspase-9 activity using Ac-DEVD-AMC and Ac-LEHD-AFC, respectively ( n = 6 animals). A , Effects of incubation with DTT, activated caspase-9 (3 U), or z-VAD-fmk on caspase-3 activity in cortex ipsilateral (right) and contralateral (left) to the FPI. DTT-treated samples exhibited significantly higher levels of caspase-3 proteolytic activity in the ipsilateral cortex compared with the contralateral cortex. B , Effects of incubation with DTT or z-VAD-fmk on caspase-9 activity in cortex ipsilateral (right) and contralateral (left) to the FPI. No significant differences in caspase-9 activity between the ipsilateral and contralateral cortex were observed with any treatment assayed.
    Recombinant Caspase 9, supplied by Millipore, used in various techniques. Bioz Stars score: 87/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Novus Biologicals anti active caspase 9
    Inhibition of the Wnt/β‐catenin signaling pathway reduces the anti‐apoptotic effect of simvastatin in the primary spinal cord neurons. (a) Western blot was utilized to detect active caspase‐3, active <t>caspase‐9,</t> Bax, and Bcl‐2 protein expression in the five experimental groups. (b–e) Lipopolysaccharide ( LPS )‐induced apoptosis increased the expression of active caspase‐3, caspase‐9, and Bax. The expression patterns were more evident after silencing the β‐catenin gene with si RNA , whereas Simv treatment significantly reduced the expression patterns of pro‐apoptotic proteins while elevating Bcl‐2 expression. The effects of Simv were reversed after silencing the β‐catenin gene with si RNA . NeuN was used as the loading control. Simv, simvastatin; ** p
    Anti Active Caspase 9, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Boster Bio anti active caspase 9
    Immunohistochemical and western-blot analysis of Apaf-1, active <t>caspase-9,</t> and active caspase-3 proteins. (A1) IHC analysis of livers in control group, (A2) IHC analysis of livers in inoculated group. The primary antibody was Apaf-1 antibody, and the positive signals were observed in the cytoplasm of hepatocyte (→); (A3) A semi-quantitative analysis of the ratio of Apaf-1 positive staining to the total field; (A4) Western-blot analysis of Apaf-1 protein in hepatocyte of control group and inoculated group gerbils; ( A5 ) A semi-quantitative analysis of relative expression of Apaf-1/β-actin. (B1) IHC analysis of livers in control group, (B2) IHC analysis of livers in inoculated group. The primary antibody was active caspase-9 antibody, and the positive signals were observed in the cytoplasm of hepatocyte (→); (B3) A semi-quantitative analysis of the ratio of active caspase-9 positive staining to the total field; (B4) Western-blot analysis of active caspase-9 protein in hepatocyte of control group and inoculated group gerbils; (B5) A semi-quantitative analysis of relative expression of active caspase-9/GAPDH. (C1) IHC analysis of livers in control group, (C2) IHC analysis of livers in inoculated group. The primary antibody was active caspase-3 antibody, and the positive signals were observed in the cytoplasm of hepatocyte (→); (C3) A semi-quantitative analysis of the ratio of active caspase-3 positive staining to the total field; (C4) Western-blot analysis of active caspase-3 protein in hepatocyte of control group and inoculated group gerbils; (C5) A semi-quantitative analysis of relative expression of active caspase-3/β-actin. ∗ p
    Anti Active Caspase 9, supplied by Boster Bio, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    MBL International caspase 9
    Characterization of endogenous AIP expression in the brain. a , RT-PCR was performed to detect AIP and <t>caspase-9</t> mRNA in various brain regions in adult rats. The primers were designed to allow the detection of both species in the same reaction. The schematic
    Caspase 9, supplied by MBL International, used in various techniques. Bioz Stars score: 88/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore fluorescence caspase 9 substrate
    Figure 4. WTp53 interacts with the p35 fragment <t>caspase-9.</t> ( A ) p53 was expressed using an IVT system, and immunoprecipitation was performed in the presence of IVT-expressed MDM2 or recombinant active caspase-9. ( B ) Apoptosis was induced with dATP and rCyt- c in S100 lysates of H1299 cells stably expressing p53R175H. Antibody pairs between DO-1 (p53) and the above was added to the lysates and anti-mouse coated (donor ) and anti-rabbit (acceptor) beads were used to detect interactions between the proteins (top panel). Fluorescence signal were measured for lysates induced for apoptosis and expressed as a ratio over signal obtained for uninduced lysates for each antibody pair (bottom left panel). Immunoblots for p53 and caspase-9 was done for the lysates used for this assay (bottom right panel).
    Fluorescence Caspase 9 Substrate, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore caspase 9
    NS3694 blocks the formation of the ∼700-kDa apoptosome complex. (A) Increasing concentrations of NS3694 were coincubated with THP.1 cell lysates (10 mg) in the presence (+) and absence (−) of 2 mM dATP-MgCl 2 for 30 min at 37°C before assaying for DEVDase activity (essentially caspases 3 and 7) and <t>caspase</t> 9 (Casp-9) and caspase 3 (Casp-3) processing as described in Materials and Methods. The positions of the procaspases (Pro) and processed forms (p35 and p37 [p35/37] and p19 and p17 [p19/p17]) are indicated to the right of the blots. NS3694 was added in a dimethyl sulfoxide solution, and control samples were incubated with equivalent amounts of the solvent, which did not inhibit caspase activation. (B) Two-milligram aliquots of control THP.1 cell lysates and dATP-activated THP.1 cell lysates in the presence and absence of NS3694 (500 μM) were fractionated on Superose 6 columns, and the fractions were analyzed for Apaf-1 and caspase 9 (Casp-9). The sizes of the various peaks were determined by using gel filtration marker proteins, and the elution positions of the ∼700-kDa and ∼1.4-MDa apoptosome complexes and aldolase (158 kDa) are indicated above the blots. Processed caspase 9 was not detected in the control samples and Ns3694-treated samples. In the dATP-activated sample, procaspase 9 (pro) was totally processed to its active subunits. The results shown are from a representative experiment, and similar data were obtained in a repeated experiment. Numbers in boxes, fraction numbers.
    Caspase 9, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Novus Biologicals caspase 9 antibody
    A utophagy-deficient mice showed increased activity of apoptosis and necroptosis. ( a ) Depletion of Atg7 increased pancreatic caspase-3 in 20-week-old Atg7 Δpan mice. Caspase-3 quantitation and representative IF microphotographs of Atg7 F/F ( n =4) and Atg7 Δpan ( n =4) pancreatic tissue stained for DAPI (blue) and Caspase-3 (red) (anti-active caspase-3 ab2302, 1/50, scale bar=50 μ m). ( b ) Reduced pancreatic Atg7 level increased the expression of caspase-8 in 12-week-old Atg7 Δpan mice. Caspase-8 quantitation and representative IF microphotographs of Atg7 F/F ( n =5) and Atg7 Δpan ( n =5) pancreatic tissue stained for DAPI (blue) and Caspase-8 (green) (anti-active caspase-8 Novusbio NB100-56116, 1/1000, scale bar=50 μ m). ( c ) Increased the expression of <t>caspase-9</t> in 12-week-old Atg7 Δpan mice. Caspase-9 quantitation and representative IF microphotographs of Atg7 F/F ( n =5) and Atg7 Δpan ( n =5) pancreatic tissue stained for DAPI (blue) and Caspase-9 (green) (anti-caspase-9 Novusbio NB100-56118, 1/1000, scale bar=50 μ m). ( d ) Reduced pancreatic Atg7 level increased the expression of Bax in 12-week-old Atg7 Δpan mice Bax quantitation and representative IF microphotographs of Atg7 F/F ( n =5) and Atg7 Δpan ( n =5) pancreatic tissue stained for DAPI (blue) and Bax (green) (anti-Bax sc-526, 1/50, scale bar=50 μ m). ( e ) Reduced pancreatic Atg7 level increased the expression of necroptotic protein Rip3 in 20-week-old Atg7 Δpan mice. Rip3 quantitation and representative IF microphotographs of Atg7 F/F ( n =5) and Atg7 Δpan ( n =5) pancreatic tissue stained for DAPI (blue) and Rip3 (green) (anti-Rip3 ab62344, 1/200, scale bar=50 μ m). ( f ) Reduced pancreatic Atg7 level increased the expression of necroptotic protein Mlkl in 20-week-old Atg7 Δpan mice. Mlkl quantitation and representative IF microphotographs of Atg7 F/F ( n =5) and Atg7 Δpan ( n =5) pancreatic tissue stained for DAPI (blue) and Mlkl (green) (anti-Mlkl ab194699, 1/100, scale bar=50 μ m). ( g ) Reduced pancreatic Atg7 level increased the expression of necroptotic protein Hmgb1 in 20-week-old Atg7 Δpan mice. Hmgb1 quantitation and representative IF microphotographs of Atg7 F/F ( n =5) and Atg7 Δpan ( n =5) pancreatic tissue stained for DAPI (blue) and Hmgb1 (red) (anti-Hmgb1 ab18256, 1/1000, scale bar=50 μ m). Data are mean±S.E.M. for the numbers of animals as indicated in the graph, * P
    Caspase 9 Antibody, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 99/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Abcam caspase 9
    Altered expression of Pitx2, Tbx5 , and Myocd in response to atrial short-term tachyarrhythmia. (a) Average relative values of transcript levels of Pitx2c , Tbx5 , and Myocd in the left atrium from paced (EXP) versus sham (CON) animals. ∗ p ≤ 0.05 ( n = 5 for each group). (b) Representative Western blots of left atrium samples from sham-operated (CON, lines 1-2) and paced (EXP, lines 3 and 4) animals and overall relative levels of the proteins as based on average values from each group. ∗ p ≤ 0.05 ( n = 5 for each group). Western blot replicates were probed with antibodies against PITX2A,B,C, TBX5, MYOCD (myocardin), CASP3 (caspase-3), CASP9 <t>(caspase-9),</t> SERCA2 (cardiac sarcoplasmic reticulum Ca(2+)-ATPase 2a), CASQ2 (cardiac calsequestrin), TNNI3 (cardiac troponin I), and GAPDH (glyceraldehyde-3-phosphate dehydrogenase). MW values of the bands detected are shown.
    Caspase 9, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 69 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc caspase 9
    AAV-mediated TRAIL overexpression induced increased expression of caspase-3, caspase-8, and <t>caspase-9,</t> and reduced expression of Bcl-2 in OSCC cells. (A) Immunofluorescence assay was carried out to detect the infection efficiency of the recombinant AAV system in the SCC25 OSCC cell line (magnification, ×400). The left panel presents light microscopy and the right field presents fluorescence microscopy. (B) Reverse transcription-quantitative polymerase chain reaction, (C) western blotting and (D) immunocytochemistry analysis (magnification, ×200) were performed to measure apoptosis-related genes expression changes in SCC25 cells with upregulation of TRAIL. Data are presented as the mean ± standard deviation. Each assay was performed independently at least three times. # P
    Caspase 9, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 224 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    R&D Systems caspase 9
    Both the extrinsic and intrinsic pathways are involved in Runx3-promoted cell apoptosis by dsRNA poly(I:C) and IAV infection. ( a ) Runx3 is required for the activation of caspase-8 and caspsas-9 by poly(I:C). BEAS-2B cells were transfected with 20 nM control siRNA (Con.), Runx3 siRNA-1 (Runx3-S1), or Runx3 siRNA-2 (Runx3-S2), grown for 72 h, then treated with high molecular weight poly(I:C) (2 μg/ml) for 0, 2 or 4 h. Equal amounts of cell lysates were subjected to Western blotting with specific antibodies against cleaved caspase-8, cleaved <t>caspase-9,</t> Runx3 or action as indicated. ( b,c ) BEAS-2B cells were infected with recombinant adenovirus containing vector alone or Runx3 and grown for 60 h. The cells were then left untreated or treated with poly(I:C) (2 μg/ml) for 4 h ( b ) or infected with (+) IAV H1N1 at MOI of 3 for 24 h ( c ) in the presence or absence of caspase-8 inhibitor Z-IETD-FMK (IETD-8, 20 μM), caspace-9 inhibitor Z-LEHD-FMK (LEHD-9, 20 μM), or a general caspase inhibitor Z-VAD-FMK (Z-VAD, 20 μM), and cell death rate was assessed as in Figure 7 . All data are means ± S.E. of triplicates. *p
    Caspase 9, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Millipore caspase 9 colorimetric activity assay kit
    Both the extrinsic and intrinsic pathways are involved in Runx3-promoted cell apoptosis by dsRNA poly(I:C) and IAV infection. ( a ) Runx3 is required for the activation of caspase-8 and caspsas-9 by poly(I:C). BEAS-2B cells were transfected with 20 nM control siRNA (Con.), Runx3 siRNA-1 (Runx3-S1), or Runx3 siRNA-2 (Runx3-S2), grown for 72 h, then treated with high molecular weight poly(I:C) (2 μg/ml) for 0, 2 or 4 h. Equal amounts of cell lysates were subjected to Western blotting with specific antibodies against cleaved caspase-8, cleaved <t>caspase-9,</t> Runx3 or action as indicated. ( b,c ) BEAS-2B cells were infected with recombinant adenovirus containing vector alone or Runx3 and grown for 60 h. The cells were then left untreated or treated with poly(I:C) (2 μg/ml) for 4 h ( b ) or infected with (+) IAV H1N1 at MOI of 3 for 24 h ( c ) in the presence or absence of caspase-8 inhibitor Z-IETD-FMK (IETD-8, 20 μM), caspace-9 inhibitor Z-LEHD-FMK (LEHD-9, 20 μM), or a general caspase inhibitor Z-VAD-FMK (Z-VAD, 20 μM), and cell death rate was assessed as in Figure 7 . All data are means ± S.E. of triplicates. *p
    Caspase 9 Colorimetric Activity Assay Kit, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam anti caspase 9
    Intracellular FGF1 WT protects SH-SY5Y cells from p53-dependent apoptosis. Stably transfected SH-SY5Y cells with either FGF1 WT or empty (mock) expression vectors were treated or not with etoposide for 16 h. ( a ) Polyclonal transfected SH-SY5Y apoptotic cells were characterized by flow cytometry after DiOC 6 (3) and PI staining (apoptotic cells are the DIOC−, PI− and size− cells). The graph represents the mean±S.E.M. of three independent experiments. Student’s t -tests were performed relative to the mock control, except where indicated ( n =3; n.s.: P > 0.05; ***: P ⩽0.001). ( b ) FGF1 expression and p53 activation (phosphorylated Ser15) were assessed by western blot in either polyclonal transfected cells (left panel) or isolated transfected cell lines (right panel). Actin detection was used as control. ( c ) FGF1 expression, <t>caspase-9</t> and -3 cleavages were assessed in polyclonal transfected cells by western blot. Actin detection was used as control
    Anti Caspase 9, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 381 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti caspase 9 antiserum
    Translocation of <t>caspase-9</t> from mitochondria to nuclei in PC12 cells during apoptosis. PC12-Puro ( a – h ) or PC12-Bcl-2 ( i – l ) cells were cultured with 25 nM Mitotracker without ( a – d ) or with ( e – l ) addition of 150 μM tamoxifen for 12 ( e – h ) or 24 hr ( i – l ). Cells were fixed, permeabilized, and incubated with anti-caspase-9 antiserum followed by FITC-conjugated secondary antibody. In some cases, the anti-caspase-9 antiserum was preadsorbed with recombinant caspase-9 protein d and l ) by using filters to selectively reveal FITC-labeled caspase-9 ( a , e , and i ), Mitotracker ( b , f , and j ), or both colors ( c , d , g , h , k , and l ). In m , PC12-Puro cells were cultured with or without 3 μM staurosporine or 150 μM tamoxifen for 12 hr. Cell lysates were prepared, were normalized for total protein content, and were analyzed by SDS/PAGE/immunoblotting by using anti-caspase-9 antiserum Bur49. Note that this antiserum preferentially reacts with the small subunit of the processed protease.
    Anti Caspase 9 Antiserum, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore caspase 9 inhibitor
    Effect of psoralidin on caspase signaling and viability of AIPC cells (A). PC-3 and DU-145 cells (70–80% confluency) were treated with 60 and 45 μM psoralidin, respectively, for 12 and 24 h, and Western blot analysis was performed using <t>caspase-9,</t> caspase-8, caspase -3 and PARP antibodies. Actin was used as the internal loading control. (B). PC-3 and DU-145 cells (70–80% confluency) were treated with psoralidin alone, caspase inhibitors alone (3 or 9) and a combination of caspase inhibitors and psoralidin, and a fluorometric assay was performed to determine caspase-3 activation. Bars represent fold increase in caspase activity in each treatment group. (C). PC-3, DU-145 and PzHPv-7 (70–80% confluency) cells were treated with varying concentrations of psoralidin, and cell viability was determined using Trypan blue assay. A dose-response curve was plotted, and each data point represents mean percentage of cell viability±SD.
    Caspase 9 Inhibitor, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 68 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems anti human caspase 9
    Role of caspases in LtxA-mediated cell death. CEM (A), Jurkat (B) and RL (C) cells were pretreated with the pancaspase inhibitor z-VAD-FMK (50 μM) for 1 hour and subsequently treated with LtxA for 24 hours. Cytotoxity was determined by flow cytometry, and the percent cell death is expressed as the sum of annexin V + /7-AAD − and annexin V + /7-AAD + cells. Data represent the average of three independent experiments. Error bars show SEM. The significance of the differences was determined using a Student’s t -test. *p≤0.05; ** p≤0.01. (D–G) Caspase-8 and -9 activation in Jurkat cells. Jurkat cells were treated with 0.2 μg/ml LtxA for various times and cleavage of caspase-8 (D, E) <t>caspase-9</t> (F) or total GAPDH levels (G) was evaluated. Solid arrows represent the procaspase form and open arrows indicate the cleavage product. D) Western blot analysis of caspase-8. Lane 1, Untreated Jurkat cells; lane 2, 2 hours LtxA; lane 3, 6 hours LtxA. E) Flow cytometric analaysis of Jurkat cells treated with 0.2 μg/ml LtxA, permeabilized, and stained for caspase-8 activation. The gray peak represents unstained cells; black, untreated cells; red, 2 hours LtxA; blue, 6 hours LtxA. F) Western blot analysis of caspase-9. First lane, untreated cells; second lane, 2 hours LtxA; third lane, 6 hours LtxA; fourth lane, 4 hours staurosporine treated cells (1 μg/ml). G) Western blot analysis of GAPDH. First lane, untreated cells; second lane, 2 hours LtxA; third lane, 6 hours LtxA. (H–J) Caspase-8 and -9 activation in RL cells. RL cells were treated with 0.2 μg/ml LtxA for various times and cleavage of caspase-8 (H) or caspase-9 (I) was evaluated by western blot analysis. Solid arrows represent the procaspase form and open arrows indicate the cleavage product. H) Western blot analysis of caspase-8. First lane, untreated cells; second lane, 3 hours LtxA; third lane, 6 hours LtxA. I) Western blot analysis of caspase-9. First lane, untreated cells; second lane, 3 hours LtxA; third lane 6 hours LtxA; fourth lane, 4 hours staurosporine treated cells (1 μg/ml). J) Western blot analysis of GAPDH. First lane, untreated cells; second lane, 3 hours LtxA; third lane, 6 hours LtxA.
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    Cell Signaling Technology Inc anti caspase 9
    Inhibition of SHH triggers cancer cell death via CDON-induced apoptosis. (A) Quantification of SHH expression by Q-RT-PCR in a panel of 45 human colorectal tumors and paired normal tissues. Data are presented as a ratio of SHH expression between tumor and normal tissue for each sample. Overexpression of SHH is considered when more than a 1.5-fold increase in the tumor is observed (indicated by the dashed line). (B) Quantification of SHH expression by Q-RT-PCR in a panel of 105 human tumors. Each tumor sample was compared to a normal paired tissue. For each type of tissue, the percentage of tumors showing an increase in SHH (considered when a more than 1.5-fold increase in expression is observed as compared to the normal tissue) is indicated. (C) Quantification of endogenous secreted SHH by ELISA assay in A549, H522, and H460 cells culture medium. H460 cell line is presented as a negative control. (D) Quantification of endogenous secreted SHH by ELISA assay in the culture medium of A549 cells transfected with scramble or SHH siRNA. (E) CDON immunofluorescence staining of A549 cells transfected with scramble or CDON siRNA using a CDON-specific antibody (in green) as described in the methods section. Nuclei were stained with Hoechst (in blue). (F) Apoptotic cell death induction as measured by TUNEL staining was quantified in A549 and H522 cells transfected with SHH siRNA alone or together with CDON siRNA. (G) <t>Caspase-9</t> activity was measured in A549 cells 18 h after transfection with SHH siRNA alone or together with CDON siRNA. (H) Nude mice were engrafted with A549 cells by subcutaneous injection of 10 million cells. When the mean tumor volume reached approximately 100 mm 3 , animals were treated twice a week by i.p. injection of scramble or SHH siRNA alone or in combination with CDON siRNA during 4 wk. Mean tumor volume and number of animals for each group are indicated. (I) Representative images of scr siRNA, SHH siRNA, or SHH siRNA+CDON siRNA-treated tumors on day 35. (J) Apoptosis quantification by caspase-3 activity assay on xenografted tumor lysates analyzed after 1 wk of treatment with siRNAs. For (H), error bars indicate s.e.m. Statistical treatment of the data was performed using a two-sided Mann–Whitney test compared to scramble siRNA-treated condition (* p
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    Bachem caspase 9
    Azathioprine induces a mitochondrial pathway of apoptosis. ( a ) Activity of caspase-3, -8, and -9 upon treatment of T cells with 6-MP. CD45RA and CD45RO T cell subsets were stimulated with antibodies to CD3 and CD28 and recombinant IL-2 for 5 days in the presence or absence of 6-MP, as indicated. There was a marked induction of <t>caspase-9</t> activity upon azathioprine treatment. A second independent experiment showed similar results (data not shown). Data on caspase-9 activity from three independent healthy blood donors are shown in the right lower panel. ( b ) Specific blockade of caspase-9 by acetyl-LEHD-CHO (Ac-LEHD-CHO) suppresses 6-MP–induced apoptosis. CD4 + T lymphocytes from the peripheral blood of healthy volunteers were stimulated with antibodies to CD3 and CD28 in the presence or absence of 6-MP, 10 μM acetyl-LEHD-CHO, and 10 μm acetyl-IETD-CHO. Although acetyl-IETD-CHO had little effect, 6-MP–induced T cell apoptosis could be suppressed by acetyl-LEHD-CHO. ( c ) Measurement of ΔΨ m in primary CD4 + T lymphocytes upon treatment with azathioprine, 6-MP, and FCCP (positive control). Peripheral blood CD4 + T cells from healthy volunteers were stimulated with antibodies to CD3 and CD28 and recombinant IL-2 and cultured in the presence or absence of azathioprine or 6-MP for 5 days as indicated. Cells were then loaded with JC-1 for 20 minutes followed by FACS analysis to determine ΔΨ m . Both azathioprine and 6-MP as well as FCCP led to a marked reduction of ΔΨ m as compared with untreated primary CD4 + T cells. One representative experiment of two is shown. Aza, azathioprine; UT, untreated.
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    GE Healthcare caspase 9
    Bcl-X L inhibits the association of Apaf-1 with <t>caspase-9.</t> 293T cells were transfected with indicated plasmids and the lysates immunoprecipitated with anti-HA antibody. ( Upper ) Western blot analysis of immunoprecipitated caspase-9 and coimmunoprecipitated Apaf-1. ( Lower ) Western blot analysis of total lysates with anti-Myc and anti-Flag antibody.
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    Enzo Biochem caspase 9 activity assay
    Functional antagonism of Smac mimetics against XIAP linker-BIR2-BIR3 in a cell-free <t>caspase-9</t> functional assay. Data shown in the figure are averages and standard deviations of duplicate wells in assay plates, and the figure is the representative of three
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    Pharmingen caspase 9
    RGDS-survivin interaction. A: Bt-RGDS interaction with cell lysate: cytoplasmic extracts were immobilized onto nitrocellulose and incubated with bt-RGDS. Total binding in the presence of labeled RGDS and nonspecific binding in the presence of an excess of unlabeled RGDS are shown. Densitometry of three different experiments and one representative experiment are reported. B: RGDS interaction with intracellular proteins was assayed by co-precipitation. BSA (control), bt-RGES (specificity control) or bt-RGDS (1 mM) were incubated for 1 h at 4°C with streptavidin-coated dynabeads, then SK-MEL-110 lysate, pre-incubated for 4 h at 4°C with an excess of unlabeled RGDS, was added and incubated overnight at 4°C. Precipitated proteins were revealed by western blotting using various antibodies (anti pro-caspase-8, anti <t>pro-caspase-9,</t> anti pro-caspase-1, anti pro-caspase-3 and anti-survivin). Total lysate was used as positive control. This experiment was carried out three times. C: Purified recombinant proteins were spotted onto nitrocellulose (0.9 μg/spot). Membrane was incubated for 4 h at RT with bt-RGDS (1 mg/ml) (total binding) in the absence or in the presence of an excess of unlabeled RGDS (10 mg/ml) (nonspecific binding). Three independent experiments were performed.
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    R&D Systems human pro caspase 9
     The time courses of proteolytic processing of pro-caspase 8 ( A ), Bid ( B ) and pro-caspase 9 ( C ) in MOLT-4 cells following intracytoplasmic delivery of CGTTA. Cells were loaded from an external concentration of 20 µM oligodeoxynucleotide and lysates were prepared at the indicated times after resuspension in fresh medium. Band intensities on western blots were determined by densitometry. The data were normalised to the values for cells treated with streptolysin O alone and each point represents the mean ± SD of three replicates.
    Human Pro Caspase 9, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Aven Tools caspase 9
    Bcr-Abl signaling does not induce phosphorylation of caspase 9. (A) Endogenous <t>caspase</t> 9 (cas-9) was immunoprecipitated from 293T cells, and in vitro kinase assays were performed with control GFP (lanes C)- or Bcr-Abl (lanes B)-expressing cell lysates supplemented with [γ- 32 P]ATP under conditions in which Bcr-Abl was autophosphorylated (bottom). Lysates were treated with recombinant active ERK (lanes E) as a control for caspase 9 phosphorylation. IgG, immunoglobulin G. (B) control GFP (lane C)- and Bcr-Abl (lane B)-expressing Rat-1 fibroblasts were radiolabeled with orthophosphate. Endogenous caspase 9 was captured with a GST fusion protein containing the CARD of Apaf-1. Caspase 9 bound to the Apaf-1 CARD was then subjected to SDS-PAGE and autoradiography or immunoblot analysis with an antibody directed against rat caspase 9. The values on the left are molecular size markers in kilodaltons.
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    Research Products Corporation caspase 9
    Identification of the Z-VAD– binding activity as caspase-2 and -9. (A) Proteins reacting with biotinylated VAD.fmk in the intermembrane space of liver mitochondria. The supernatant of control mitochondria (lane 1) or of Atr-treated mitochondria (lanes 2 and 3), the flow-through of the MiniS column (see Fig. 2 and Fig. 3 A; lanes 4 and 5), purified AIF (lane 6), or the Z-VAD.afc–cleaving activity eluting at 280 mM from the MiniQ column (see Fig. 2 and Fig. 4 A; lanes 7 and 8) were allowed to react with biotinylated VAD.fmk, either without pretreatment (lanes 1, 2, 4, 6, and 7) or after preincubation with Z-VAD.fmk (lanes 3, 5, and 8). Note that Z-VAD.fmk has been added to mitochondria before Atr (line 3). In addition, the proteins reacting with biotinylated VAD.fmk retained on an avidin column were purified (lane 9). These proteins, which contained approximately similar levels of Z-VAD.afc–cleaving activity (10 U) or ∼100 ng purified protein (lane 6) were separated by SDS-PAGE, blotted onto nitrocellulose, and subjected to the detection of biotinylated VAD.fmk using an avidin-based detection system. (B and C) The same blot as in A was subjected to immunodetection with antibodies specific for caspase-2 (B) or -9 (C). (D and E) Mitochondria from different organs were purified and cultured for 30 min in the presence or absence of 5 mM Atr, followed by immunoblot detection of caspase-2 (D) or -9 (E). Results are representative of two to four independent experiments. (F and G) Specificity control of caspase-2– and <t>caspase-9–specific</t> antisera. Recombinant caspase-2 (lane 1), -3 (lane 2), or -9 (lane 3) was immunoblotted (100 ng/lane), followed by immunodetection with the caspase-2 (F) or caspase-9 (G)–specific antibody. Similarly, caspase-2– and caspase-9–specific antibodies fail to recognize caspase-6 and -7 (not shown).
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    Enzo Biochem caspase 9
    A novel pathway of Tom20-Bax-caspase-GSDME upon iron stimulation. In melanoma cells, iron-elevated ROS causes the oxidation and oligomerization of Tom20. Oxidized Tom20 induces Bax translocation to mitochondria, which facilitates cytochrome c release to cytosol. Once released, cytochrome c activates <t>caspase-9,</t> which then activates caspase-3. This caspase-3 activation further cleaves GSDME, and eventually triggers cell swelling and LDH release
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    Image Search Results


    Phosphorylation of murine caspase-9 CK2 decreases its susceptibility to cleavage by active caspase-8. A , purified caspase-9-FLAG was incubated with [γ- 32 P]ATP in kinase buffer in the presence or absence of purified recombinant CK2. Aliquots of the kinase reactions above were used as the source of substrate in cleavage assays using active, recombinant caspase-8 protein. Attenuated reactions were SDS-PAGE-resolved, transferred to nitrocellulose, and autoradiographed to visualize phosphorylated caspase-9 ( top panel ). The same membrane was then immunoblotted with an antibody against caspase-9 (Stressgen) to visualize total full-length and cleaved protein and determine relative cleavage by caspase-8 of unphosphorylated and phosphorylated substrates. B, in vitro translated [ 35 S]methionine-labeled mC-9 was used as substrate for phosphorylation by recombinant CK2. Kinase reactions were performed in the presence of cold ATP with or without CK2, following which caspase-8 cleavage reactions were carried out on aliquots of the kinase reactions. The results were visualized by autoradiography following resolution by SDS-PAGE. C , alternatively the kinase reactions were diluted in rehydration buffer and resolved by isoelectric focusing ( IEF , pH range 5–8) in the first dimension, followed by electrophoresis in the second dimension using an 8–16% gradient.

    Journal: The Journal of Biological Chemistry

    Article Title: Phosphorylation of Murine Caspase-9 by the Protein Kinase Casein Kinase 2 Regulates Its Cleavage by Caspase-8 *Phosphorylation of Murine Caspase-9 by the Protein Kinase Casein Kinase 2 Regulates Its Cleavage by Caspase-8 * S⃞

    doi: 10.1074/jbc.M802846200

    Figure Lengend Snippet: Phosphorylation of murine caspase-9 CK2 decreases its susceptibility to cleavage by active caspase-8. A , purified caspase-9-FLAG was incubated with [γ- 32 P]ATP in kinase buffer in the presence or absence of purified recombinant CK2. Aliquots of the kinase reactions above were used as the source of substrate in cleavage assays using active, recombinant caspase-8 protein. Attenuated reactions were SDS-PAGE-resolved, transferred to nitrocellulose, and autoradiographed to visualize phosphorylated caspase-9 ( top panel ). The same membrane was then immunoblotted with an antibody against caspase-9 (Stressgen) to visualize total full-length and cleaved protein and determine relative cleavage by caspase-8 of unphosphorylated and phosphorylated substrates. B, in vitro translated [ 35 S]methionine-labeled mC-9 was used as substrate for phosphorylation by recombinant CK2. Kinase reactions were performed in the presence of cold ATP with or without CK2, following which caspase-8 cleavage reactions were carried out on aliquots of the kinase reactions. The results were visualized by autoradiography following resolution by SDS-PAGE. C , alternatively the kinase reactions were diluted in rehydration buffer and resolved by isoelectric focusing ( IEF , pH range 5–8) in the first dimension, followed by electrophoresis in the second dimension using an 8–16% gradient.

    Article Snippet: After blocking in 1% bovine serum albumin, the permeabilized cells were incubated with anti-caspase-9 antibody for 1 h, then washed, and incubated with Alexa 488-tagged goat anti-mouse IgG (Invitrogen).

    Techniques: Purification, Incubation, Recombinant, SDS Page, In Vitro, Labeling, Autoradiography, Electrofocusing, Electrophoresis

    Simplified model of mitochondrial and TNF receptor-activated apoptotic pathways in murine cells. The kinase CK2 controls activation of caspase-9 in receptor-activated apoptotic pathways where it is processed by initiator caspase-8 but not in mitochondrial pathways where it is activated by autoprocessing.

    Journal: The Journal of Biological Chemistry

    Article Title: Phosphorylation of Murine Caspase-9 by the Protein Kinase Casein Kinase 2 Regulates Its Cleavage by Caspase-8 *Phosphorylation of Murine Caspase-9 by the Protein Kinase Casein Kinase 2 Regulates Its Cleavage by Caspase-8 * S⃞

    doi: 10.1074/jbc.M802846200

    Figure Lengend Snippet: Simplified model of mitochondrial and TNF receptor-activated apoptotic pathways in murine cells. The kinase CK2 controls activation of caspase-9 in receptor-activated apoptotic pathways where it is processed by initiator caspase-8 but not in mitochondrial pathways where it is activated by autoprocessing.

    Article Snippet: After blocking in 1% bovine serum albumin, the permeabilized cells were incubated with anti-caspase-9 antibody for 1 h, then washed, and incubated with Alexa 488-tagged goat anti-mouse IgG (Invitrogen).

    Techniques: Activation Assay

    CK2-specific inhibitor accelerates caspase-9 activation, and phosphatase inhibitor delays caspase-9 processing in response to TNF-α/CHX. A , FL5.12 cells were exposed to TNF-α/CHX for 6 h following incubation in medium containing either the CK2-specific inhibitor TBB or vehicle (dimethyl sulfoxide, DMSO ) for 3 h. The graph shows the percentage of viability of cells harvested at 0, 2, 4, and 6 h following the addition of ligand (mean and standard error, n = 3). B , graph showing caspase-9 activity in Neo and Bcl-x L cells following exposure to TNF-α/CHX in the presence or absence of TBB, as determined by colorimetric substrate (LEHD-pNA) cleavage assays. C , Western blots of lysates (50 μg of protein) of 0-, 2-, and 4-h time points from the above experiment, showing processed caspase-8, p10 fragment ( top panel of each set), full-length and cleaved caspase-9 ( center ), and loading control, actin ( bottom ). The membranes were sequentially stripped and reprobed.

    Journal: The Journal of Biological Chemistry

    Article Title: Phosphorylation of Murine Caspase-9 by the Protein Kinase Casein Kinase 2 Regulates Its Cleavage by Caspase-8 *Phosphorylation of Murine Caspase-9 by the Protein Kinase Casein Kinase 2 Regulates Its Cleavage by Caspase-8 * S⃞

    doi: 10.1074/jbc.M802846200

    Figure Lengend Snippet: CK2-specific inhibitor accelerates caspase-9 activation, and phosphatase inhibitor delays caspase-9 processing in response to TNF-α/CHX. A , FL5.12 cells were exposed to TNF-α/CHX for 6 h following incubation in medium containing either the CK2-specific inhibitor TBB or vehicle (dimethyl sulfoxide, DMSO ) for 3 h. The graph shows the percentage of viability of cells harvested at 0, 2, 4, and 6 h following the addition of ligand (mean and standard error, n = 3). B , graph showing caspase-9 activity in Neo and Bcl-x L cells following exposure to TNF-α/CHX in the presence or absence of TBB, as determined by colorimetric substrate (LEHD-pNA) cleavage assays. C , Western blots of lysates (50 μg of protein) of 0-, 2-, and 4-h time points from the above experiment, showing processed caspase-8, p10 fragment ( top panel of each set), full-length and cleaved caspase-9 ( center ), and loading control, actin ( bottom ). The membranes were sequentially stripped and reprobed.

    Article Snippet: After blocking in 1% bovine serum albumin, the permeabilized cells were incubated with anti-caspase-9 antibody for 1 h, then washed, and incubated with Alexa 488-tagged goat anti-mouse IgG (Invitrogen).

    Techniques: Activation Assay, Incubation, Activity Assay, Western Blot

    Caspase-9 dephosphorylation and cleavage are both necessary and sufficient for an apoptotic response to TNF-α. A , Bcl-x L FL5.12 cells were labeled with [ 32 P]orthophosphate (0.5 μCi) for 6 h. Three hours following the addition of radiolabel, the culture medium was supplemented with TNF-αCHX or CHX alone. Endogenous caspase-9 was immunoprecipitated, SDS-PAGE-resolved, transferred to nitrocellulose, and visualized, first by autoradiography ( top panel ), and then by Western blotting ( lower panel ) with an antibody against caspase-9 (AAM-139, Stressgen) that recognizes both full-length and processed protein. B , caspase-9 -/- fibroblasts were transiently transfected with empty vector, wild type caspase-9, LDAD, or SEPA constructs in chamber slides and either incubated with TNF-αCHX (or CHX alone, not shown) 36 h post-transfection. The cells were fixed and stained 4 h later and observed using an Olympus Fluoview 1000 multiphoton confocal microscope. C , quantification of data from the transfection experiments. The y axis indicates percent caspase-9 expressing green fluorescent cells with condensed (apoptotic) nuclei (mean and standard error, n = 3).

    Journal: The Journal of Biological Chemistry

    Article Title: Phosphorylation of Murine Caspase-9 by the Protein Kinase Casein Kinase 2 Regulates Its Cleavage by Caspase-8 *Phosphorylation of Murine Caspase-9 by the Protein Kinase Casein Kinase 2 Regulates Its Cleavage by Caspase-8 * S⃞

    doi: 10.1074/jbc.M802846200

    Figure Lengend Snippet: Caspase-9 dephosphorylation and cleavage are both necessary and sufficient for an apoptotic response to TNF-α. A , Bcl-x L FL5.12 cells were labeled with [ 32 P]orthophosphate (0.5 μCi) for 6 h. Three hours following the addition of radiolabel, the culture medium was supplemented with TNF-αCHX or CHX alone. Endogenous caspase-9 was immunoprecipitated, SDS-PAGE-resolved, transferred to nitrocellulose, and visualized, first by autoradiography ( top panel ), and then by Western blotting ( lower panel ) with an antibody against caspase-9 (AAM-139, Stressgen) that recognizes both full-length and processed protein. B , caspase-9 -/- fibroblasts were transiently transfected with empty vector, wild type caspase-9, LDAD, or SEPA constructs in chamber slides and either incubated with TNF-αCHX (or CHX alone, not shown) 36 h post-transfection. The cells were fixed and stained 4 h later and observed using an Olympus Fluoview 1000 multiphoton confocal microscope. C , quantification of data from the transfection experiments. The y axis indicates percent caspase-9 expressing green fluorescent cells with condensed (apoptotic) nuclei (mean and standard error, n = 3).

    Article Snippet: After blocking in 1% bovine serum albumin, the permeabilized cells were incubated with anti-caspase-9 antibody for 1 h, then washed, and incubated with Alexa 488-tagged goat anti-mouse IgG (Invitrogen).

    Techniques: De-Phosphorylation Assay, Labeling, Immunoprecipitation, SDS Page, Autoradiography, Western Blot, Transfection, Plasmid Preparation, Construct, Incubation, Staining, Microscopy, Expressing

    Purified recombinant CK2 phosphorylates murine caspase-9 at Ser 348 in vitro . A , amino acid sequence in murine caspase-9 between the active site, QACGG ( * ), and the caspase-3 processing site. Arrows show the auto-processing and primary caspase-8 cleavage site, SEPD, and the caspase-3 site, DQLD. The filled black circle shows serine 348 predicted to be the target residue within a strong CK2 consensus motif ( underlined ). B–D, in vitro translated mC-9 protein was immunoprecipitated with anti-caspase-9 antibody and incubated in kinase buffer in the presence or absence of purified CK2. B , phosphorylation of mC-9 in the presence of CK2 inhibitors ( lanes 3–5 ): DRB, heparin, and apigenin. C, top panel , CK2 phosphorylation of mC-9 and the LDAD mutant in the presence or absence of DRB in vitro. Lower panel , immunoprecipitation ( IP )/Western of cold in vitro translated mC-9 and LDAD. D , CK2 phosphorylation of mC-9 and mutants AEPD and LDVD in vitro .

    Journal: The Journal of Biological Chemistry

    Article Title: Phosphorylation of Murine Caspase-9 by the Protein Kinase Casein Kinase 2 Regulates Its Cleavage by Caspase-8 *Phosphorylation of Murine Caspase-9 by the Protein Kinase Casein Kinase 2 Regulates Its Cleavage by Caspase-8 * S⃞

    doi: 10.1074/jbc.M802846200

    Figure Lengend Snippet: Purified recombinant CK2 phosphorylates murine caspase-9 at Ser 348 in vitro . A , amino acid sequence in murine caspase-9 between the active site, QACGG ( * ), and the caspase-3 processing site. Arrows show the auto-processing and primary caspase-8 cleavage site, SEPD, and the caspase-3 site, DQLD. The filled black circle shows serine 348 predicted to be the target residue within a strong CK2 consensus motif ( underlined ). B–D, in vitro translated mC-9 protein was immunoprecipitated with anti-caspase-9 antibody and incubated in kinase buffer in the presence or absence of purified CK2. B , phosphorylation of mC-9 in the presence of CK2 inhibitors ( lanes 3–5 ): DRB, heparin, and apigenin. C, top panel , CK2 phosphorylation of mC-9 and the LDAD mutant in the presence or absence of DRB in vitro. Lower panel , immunoprecipitation ( IP )/Western of cold in vitro translated mC-9 and LDAD. D , CK2 phosphorylation of mC-9 and mutants AEPD and LDVD in vitro .

    Article Snippet: After blocking in 1% bovine serum albumin, the permeabilized cells were incubated with anti-caspase-9 antibody for 1 h, then washed, and incubated with Alexa 488-tagged goat anti-mouse IgG (Invitrogen).

    Techniques: Purification, Recombinant, In Vitro, Sequencing, Immunoprecipitation, Incubation, Mutagenesis, Western Blot

    Exogenous cytochrome c /dATP fails to promote caspase-3 processing within platelet lysates. Cytosolic extracts were treated with cytochrome c and dATP or buffer alone and incubated at 37°C. (a) Western blot analysis of platelet lysates revealed a failure of cytochrome c /dATP to promote the processing of caspase-3. (b) By contrast, Jurkat T cell lysates showed progressive processing of the enzyme to the proteolytically active p17/p12 fragments. (c) On addition of human recombinant caspase-9 and cytochrome c /dATP to fresh platelet lysates, derived from the same preparation used above, caspase-3 was now processed to its active p17/p12 subunits. Each Western blot had 50 μg of protein loaded per lane and was repeated twice.

    Journal: The Journal of Cell Biology

    Article Title: Compartmentalized megakaryocyte death generates functional platelets committed to caspase-independent death

    doi: 10.1083/jcb.200210111

    Figure Lengend Snippet: Exogenous cytochrome c /dATP fails to promote caspase-3 processing within platelet lysates. Cytosolic extracts were treated with cytochrome c and dATP or buffer alone and incubated at 37°C. (a) Western blot analysis of platelet lysates revealed a failure of cytochrome c /dATP to promote the processing of caspase-3. (b) By contrast, Jurkat T cell lysates showed progressive processing of the enzyme to the proteolytically active p17/p12 fragments. (c) On addition of human recombinant caspase-9 and cytochrome c /dATP to fresh platelet lysates, derived from the same preparation used above, caspase-3 was now processed to its active p17/p12 subunits. Each Western blot had 50 μg of protein loaded per lane and was repeated twice.

    Article Snippet: Cell free apoptosis was initiated by addition of 10 μg/ml cytochrome c and 1 mM dATP, and in some experiments, 0.1 U/ml human recombinant caspase-9 (CHEMICON International), followed by incubation at 37°C.

    Techniques: Incubation, Western Blot, Recombinant, Derivative Assay

    Platelets contain APAF-1, but do not contain caspase-9. (a) Whole-cell lysates prepared from fresh platelets (i), Jurkat T cells (ii), and MEG-01 MKs (iii) were probed with an anti-APAF-1 pAb. All cells contained an immunodetectable protein with an apparent molecular mass of ∼130 kD, consistent with it being APAF-1. (b) Whole-cell lysates prepared from Jurkat T cells (i), fresh platelets (ii and iii), MEG-01 MKs (iv), and SET-2 MKs (v) were probed by Western blot for caspase-9 with a pAb that recognized both pro- and activated forms. A major band was detected with an apparent molecular mass approximating that of caspase-9 (48 kD) within Jurkat T cells and MKs. The Western blot was stripped and reprobed for β-actin, where β-actin accounts for 30% of platelet protein.

    Journal: The Journal of Cell Biology

    Article Title: Compartmentalized megakaryocyte death generates functional platelets committed to caspase-independent death

    doi: 10.1083/jcb.200210111

    Figure Lengend Snippet: Platelets contain APAF-1, but do not contain caspase-9. (a) Whole-cell lysates prepared from fresh platelets (i), Jurkat T cells (ii), and MEG-01 MKs (iii) were probed with an anti-APAF-1 pAb. All cells contained an immunodetectable protein with an apparent molecular mass of ∼130 kD, consistent with it being APAF-1. (b) Whole-cell lysates prepared from Jurkat T cells (i), fresh platelets (ii and iii), MEG-01 MKs (iv), and SET-2 MKs (v) were probed by Western blot for caspase-9 with a pAb that recognized both pro- and activated forms. A major band was detected with an apparent molecular mass approximating that of caspase-9 (48 kD) within Jurkat T cells and MKs. The Western blot was stripped and reprobed for β-actin, where β-actin accounts for 30% of platelet protein.

    Article Snippet: Cell free apoptosis was initiated by addition of 10 μg/ml cytochrome c and 1 mM dATP, and in some experiments, 0.1 U/ml human recombinant caspase-9 (CHEMICON International), followed by incubation at 37°C.

    Techniques: Western Blot

    Caspase inhibition via cysteinyl oxidation in FPI affects caspase-3 preferentially to caspase-9. Cortical lysates were harvested at 24 h from FPI injured animals (2.4 atm) and assayed for caspase-3 and caspase-9 activity using Ac-DEVD-AMC and Ac-LEHD-AFC, respectively ( n = 6 animals). A , Effects of incubation with DTT, activated caspase-9 (3 U), or z-VAD-fmk on caspase-3 activity in cortex ipsilateral (right) and contralateral (left) to the FPI. DTT-treated samples exhibited significantly higher levels of caspase-3 proteolytic activity in the ipsilateral cortex compared with the contralateral cortex. B , Effects of incubation with DTT or z-VAD-fmk on caspase-9 activity in cortex ipsilateral (right) and contralateral (left) to the FPI. No significant differences in caspase-9 activity between the ipsilateral and contralateral cortex were observed with any treatment assayed.

    Journal: The Journal of Neuroscience

    Article Title: Inhibition of Caspase-Mediated Apoptosis by Peroxynitrite in Traumatic Brain Injury

    doi: 10.1523/JNEUROSCI.3507-06.2006

    Figure Lengend Snippet: Caspase inhibition via cysteinyl oxidation in FPI affects caspase-3 preferentially to caspase-9. Cortical lysates were harvested at 24 h from FPI injured animals (2.4 atm) and assayed for caspase-3 and caspase-9 activity using Ac-DEVD-AMC and Ac-LEHD-AFC, respectively ( n = 6 animals). A , Effects of incubation with DTT, activated caspase-9 (3 U), or z-VAD-fmk on caspase-3 activity in cortex ipsilateral (right) and contralateral (left) to the FPI. DTT-treated samples exhibited significantly higher levels of caspase-3 proteolytic activity in the ipsilateral cortex compared with the contralateral cortex. B , Effects of incubation with DTT or z-VAD-fmk on caspase-9 activity in cortex ipsilateral (right) and contralateral (left) to the FPI. No significant differences in caspase-9 activity between the ipsilateral and contralateral cortex were observed with any treatment assayed.

    Article Snippet: After 1 h incubation at 37°C, recombinant active caspase-9 (Sigma) was added to the system, and caspase-3 activity was assessed using the fluorogenic Ac-DEVD-AMC assay (see Materials and Methods).

    Techniques: Inhibition, Activity Assay, Incubation

    In vitro inhibition of caspase-9 activity by AMVIAP. Human caspase-9 activity was assayed in vitro with Ac-LEHD-AMC as a substrate. Caspase-9 was mixed with either GST at 2 μM (○), XIAP at 500 nM (+), and AMVIAP at 500 nM (•) or 5,000 nM (5 μM) (▴). Activity was estimated by LEHD-AMC cleavage. Each data series represents fluorescence emission (490 nm) at 1-min intervals for 120 min.

    Journal: Journal of Virology

    Article Title: Functional Analysis of the Inhibitor of Apoptosis (iap) Gene Carried by the Entomopoxvirus of Amsacta moorei

    doi: 10.1128/JVI.79.4.2335-2345.2005

    Figure Lengend Snippet: In vitro inhibition of caspase-9 activity by AMVIAP. Human caspase-9 activity was assayed in vitro with Ac-LEHD-AMC as a substrate. Caspase-9 was mixed with either GST at 2 μM (○), XIAP at 500 nM (+), and AMVIAP at 500 nM (•) or 5,000 nM (5 μM) (▴). Activity was estimated by LEHD-AMC cleavage. Each data series represents fluorescence emission (490 nm) at 1-min intervals for 120 min.

    Article Snippet: Caspase-9 reactions were performed in 96-well plates containing caspase reaction buffer supplemented with 0.4 mM Ac-LEHD-AMC (Biomol) and 200 U of recombinant active caspase-9 (100 U/μl; Biomol) in a total volume of 100 μl.

    Techniques: In Vitro, Inhibition, Activity Assay, Fluorescence

    In vitro inhibition of caspase-3 activity by AMVIAP. Human caspase-9 activity was assayed in vitro with Ac-DEVD-AMC as the substrate. Caspase-3 was mixed with either, in the absence of recombinant IAPs (▪), GST at 2 μM (•), GST-XIAP at 4.1 μg (500 nM final) (+), or GST-AMVIAP at 2.9 μg (500 nM) (○). Activity was estimated by DEVD-AMC cleavage. Each data series represents fluorescence emission (490 nm) at 1-min intervals for 120 min.

    Journal: Journal of Virology

    Article Title: Functional Analysis of the Inhibitor of Apoptosis (iap) Gene Carried by the Entomopoxvirus of Amsacta moorei

    doi: 10.1128/JVI.79.4.2335-2345.2005

    Figure Lengend Snippet: In vitro inhibition of caspase-3 activity by AMVIAP. Human caspase-9 activity was assayed in vitro with Ac-DEVD-AMC as the substrate. Caspase-3 was mixed with either, in the absence of recombinant IAPs (▪), GST at 2 μM (•), GST-XIAP at 4.1 μg (500 nM final) (+), or GST-AMVIAP at 2.9 μg (500 nM) (○). Activity was estimated by DEVD-AMC cleavage. Each data series represents fluorescence emission (490 nm) at 1-min intervals for 120 min.

    Article Snippet: Caspase-9 reactions were performed in 96-well plates containing caspase reaction buffer supplemented with 0.4 mM Ac-LEHD-AMC (Biomol) and 200 U of recombinant active caspase-9 (100 U/μl; Biomol) in a total volume of 100 μl.

    Techniques: In Vitro, Inhibition, Activity Assay, Recombinant, Fluorescence

    Inhibition assay of wild-type and designed XIAPs on caspase-9 proteolytic activity by the Caspase-Glo® 9 Assay kit from Promega. The percent inhibitions are converted from the relative light units at different concentration of XIAP proteins. Lines connect data points to guide the eye.

    Journal: Journal of molecular biology

    Article Title: Changing the Apoptosis Pathway through Evolutionary Protein Design

    doi: 10.1016/j.jmb.2018.12.016

    Figure Lengend Snippet: Inhibition assay of wild-type and designed XIAPs on caspase-9 proteolytic activity by the Caspase-Glo® 9 Assay kit from Promega. The percent inhibitions are converted from the relative light units at different concentration of XIAP proteins. Lines connect data points to guide the eye.

    Article Snippet: The enzymatic activity of active recombinant caspase-9 (Enzo Life Sciences) was evaluated by the Caspase-Glo® 9 Assay kit from Promega, in which catalysis of a substrate by caspase-9 releases a substrate for luciferase (aminoluciferin), resulting in the luciferase reaction and a detectable luminescence emission in vitro .

    Techniques: Inhibition, Activity Assay, Concentration Assay

    Caspase inhibition via cysteinyl oxidation in FPI affects caspase-3 preferentially to caspase-9. Cortical lysates were harvested at 24 h from FPI injured animals (2.4 atm) and assayed for caspase-3 and caspase-9 activity using Ac-DEVD-AMC and Ac-LEHD-AFC, respectively ( n = 6 animals). A , Effects of incubation with DTT, activated caspase-9 (3 U), or z-VAD-fmk on caspase-3 activity in cortex ipsilateral (right) and contralateral (left) to the FPI. DTT-treated samples exhibited significantly higher levels of caspase-3 proteolytic activity in the ipsilateral cortex compared with the contralateral cortex. B , Effects of incubation with DTT or z-VAD-fmk on caspase-9 activity in cortex ipsilateral (right) and contralateral (left) to the FPI. No significant differences in caspase-9 activity between the ipsilateral and contralateral cortex were observed with any treatment assayed.

    Journal: The Journal of Neuroscience

    Article Title: Inhibition of Caspase-Mediated Apoptosis by Peroxynitrite in Traumatic Brain Injury

    doi: 10.1523/JNEUROSCI.3507-06.2006

    Figure Lengend Snippet: Caspase inhibition via cysteinyl oxidation in FPI affects caspase-3 preferentially to caspase-9. Cortical lysates were harvested at 24 h from FPI injured animals (2.4 atm) and assayed for caspase-3 and caspase-9 activity using Ac-DEVD-AMC and Ac-LEHD-AFC, respectively ( n = 6 animals). A , Effects of incubation with DTT, activated caspase-9 (3 U), or z-VAD-fmk on caspase-3 activity in cortex ipsilateral (right) and contralateral (left) to the FPI. DTT-treated samples exhibited significantly higher levels of caspase-3 proteolytic activity in the ipsilateral cortex compared with the contralateral cortex. B , Effects of incubation with DTT or z-VAD-fmk on caspase-9 activity in cortex ipsilateral (right) and contralateral (left) to the FPI. No significant differences in caspase-9 activity between the ipsilateral and contralateral cortex were observed with any treatment assayed.

    Article Snippet: Recombinant caspase-9 (Calbiochem; 1 U) was diluted in M-PER to a final concentration of 0.01 U/μl and incubated with peroxynitrite for 1 h at 37°C.

    Techniques: Inhibition, Activity Assay, Incubation

    Inhibition of the Wnt/β‐catenin signaling pathway reduces the anti‐apoptotic effect of simvastatin in the primary spinal cord neurons. (a) Western blot was utilized to detect active caspase‐3, active caspase‐9, Bax, and Bcl‐2 protein expression in the five experimental groups. (b–e) Lipopolysaccharide ( LPS )‐induced apoptosis increased the expression of active caspase‐3, caspase‐9, and Bax. The expression patterns were more evident after silencing the β‐catenin gene with si RNA , whereas Simv treatment significantly reduced the expression patterns of pro‐apoptotic proteins while elevating Bcl‐2 expression. The effects of Simv were reversed after silencing the β‐catenin gene with si RNA . NeuN was used as the loading control. Simv, simvastatin; ** p

    Journal: Journal of Neurochemistry

    Article Title: Simvastatin inhibits neural cell apoptosis and promotes locomotor recovery via activation of Wnt/β‐catenin signaling pathway after spinal cord injury

    doi: 10.1111/jnc.13382

    Figure Lengend Snippet: Inhibition of the Wnt/β‐catenin signaling pathway reduces the anti‐apoptotic effect of simvastatin in the primary spinal cord neurons. (a) Western blot was utilized to detect active caspase‐3, active caspase‐9, Bax, and Bcl‐2 protein expression in the five experimental groups. (b–e) Lipopolysaccharide ( LPS )‐induced apoptosis increased the expression of active caspase‐3, caspase‐9, and Bax. The expression patterns were more evident after silencing the β‐catenin gene with si RNA , whereas Simv treatment significantly reduced the expression patterns of pro‐apoptotic proteins while elevating Bcl‐2 expression. The effects of Simv were reversed after silencing the β‐catenin gene with si RNA . NeuN was used as the loading control. Simv, simvastatin; ** p

    Article Snippet: The membranes were blocked for 2 h at 23 ± 2°C and then incubated overnight at 4°C with the following primary antibodies: anti‐p‐LRP‐6 (1 : 500; Cell Signaling Technology, Inc., Boston, USA), anti‐β‐catenin (1 : 500; Cell Signaling Technology, Inc.), anti‐p‐β‐catenin (Ser33/37/Thr41) (1 : 500; Cell Signaling Technology, Inc.), anti‐active caspase‐3 (1 : 1000; Novus Biologicals, Littleton, CO, USA), anti‐active caspase‐9 (1 : 1000; Novus Biologicals), anti‐Bcl‐2 (1 : 1000; Novus Biologicals), anti‐Bax (1 : 1000; Novus Biologicals), anti‐NeuN antibody (1 : 300; Abcam, Cambridge, UK), and anti‐β‐actin (1 : 1000; Abcam).

    Techniques: Inhibition, Western Blot, Expressing

    Anti‐apoptotic effect of simvastatin in the primary spinal cord neurons was reduced following the suppression of the Wnt/β‐catenin signaling pathway. (a) The expression levels of Bcl‐2, Bax, active caspase‐9, and caspase‐3 in primary spinal cord neurons were detected using western blot analysis. (b–e) Simv observably restrained the apoptosis of spinal cord neurons after lipopolysaccharide ( LPS ) treatment, whereas the beneficial anti‐apoptotic effect of Simv on spinal cord neurons was destroyed after the inhibition of the Wnt/β‐catenin signaling pathway by the Wnt antagonist XAV 939. NeuN was used as the loading control. Simv, simvastatin; * p

    Journal: Journal of Neurochemistry

    Article Title: Simvastatin inhibits neural cell apoptosis and promotes locomotor recovery via activation of Wnt/β‐catenin signaling pathway after spinal cord injury

    doi: 10.1111/jnc.13382

    Figure Lengend Snippet: Anti‐apoptotic effect of simvastatin in the primary spinal cord neurons was reduced following the suppression of the Wnt/β‐catenin signaling pathway. (a) The expression levels of Bcl‐2, Bax, active caspase‐9, and caspase‐3 in primary spinal cord neurons were detected using western blot analysis. (b–e) Simv observably restrained the apoptosis of spinal cord neurons after lipopolysaccharide ( LPS ) treatment, whereas the beneficial anti‐apoptotic effect of Simv on spinal cord neurons was destroyed after the inhibition of the Wnt/β‐catenin signaling pathway by the Wnt antagonist XAV 939. NeuN was used as the loading control. Simv, simvastatin; * p

    Article Snippet: The membranes were blocked for 2 h at 23 ± 2°C and then incubated overnight at 4°C with the following primary antibodies: anti‐p‐LRP‐6 (1 : 500; Cell Signaling Technology, Inc., Boston, USA), anti‐β‐catenin (1 : 500; Cell Signaling Technology, Inc.), anti‐p‐β‐catenin (Ser33/37/Thr41) (1 : 500; Cell Signaling Technology, Inc.), anti‐active caspase‐3 (1 : 1000; Novus Biologicals, Littleton, CO, USA), anti‐active caspase‐9 (1 : 1000; Novus Biologicals), anti‐Bcl‐2 (1 : 1000; Novus Biologicals), anti‐Bax (1 : 1000; Novus Biologicals), anti‐NeuN antibody (1 : 300; Abcam, Cambridge, UK), and anti‐β‐actin (1 : 1000; Abcam).

    Techniques: Expressing, Western Blot, Inhibition

    Neuronal apoptosis is inhibited by simvastatin after spinal cord injury ( SCI ). (a) The expression levels of Bcl‐2, Bax, active caspase‐3, and caspase‐9 in the spinal cord neurons were detected using western blot analysis at 3, 7, and 14 days after SCI . (b) Bcl‐2 expression decreased, whereas (c) Bax, (d) active caspase‐9, and (e) active caspase‐3 levels increased in the vehicle‐treated rats compared with the sham‐treated rats after SCI . However, Simv treatment significantly reduced the expression of Bax, active caspase‐9, and active caspase‐3, and increased the expression of Bcl‐2, compared with vehicle treatment. NeuN was used as the loading control. Simv, simvastatin; * p

    Journal: Journal of Neurochemistry

    Article Title: Simvastatin inhibits neural cell apoptosis and promotes locomotor recovery via activation of Wnt/β‐catenin signaling pathway after spinal cord injury

    doi: 10.1111/jnc.13382

    Figure Lengend Snippet: Neuronal apoptosis is inhibited by simvastatin after spinal cord injury ( SCI ). (a) The expression levels of Bcl‐2, Bax, active caspase‐3, and caspase‐9 in the spinal cord neurons were detected using western blot analysis at 3, 7, and 14 days after SCI . (b) Bcl‐2 expression decreased, whereas (c) Bax, (d) active caspase‐9, and (e) active caspase‐3 levels increased in the vehicle‐treated rats compared with the sham‐treated rats after SCI . However, Simv treatment significantly reduced the expression of Bax, active caspase‐9, and active caspase‐3, and increased the expression of Bcl‐2, compared with vehicle treatment. NeuN was used as the loading control. Simv, simvastatin; * p

    Article Snippet: The membranes were blocked for 2 h at 23 ± 2°C and then incubated overnight at 4°C with the following primary antibodies: anti‐p‐LRP‐6 (1 : 500; Cell Signaling Technology, Inc., Boston, USA), anti‐β‐catenin (1 : 500; Cell Signaling Technology, Inc.), anti‐p‐β‐catenin (Ser33/37/Thr41) (1 : 500; Cell Signaling Technology, Inc.), anti‐active caspase‐3 (1 : 1000; Novus Biologicals, Littleton, CO, USA), anti‐active caspase‐9 (1 : 1000; Novus Biologicals), anti‐Bcl‐2 (1 : 1000; Novus Biologicals), anti‐Bax (1 : 1000; Novus Biologicals), anti‐NeuN antibody (1 : 300; Abcam, Cambridge, UK), and anti‐β‐actin (1 : 1000; Abcam).

    Techniques: Expressing, Western Blot

    Relative levels of Fas ( A ), FADD ( B ), caspase-3 ( C ), caspase-8 ( D ), and caspase-9 ( E ) mRNA transcripts evaluated by real-time PCR as well as caspase-3 ( F ), caspase-8 ( G ), and caspase-9 ( H ) activities. Data were shown in mean ± SEM.

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Interferon-? Exacerbates Dry Eye-Induced Apoptosis in Conjunctiva through Dual Apoptotic Pathways

    doi: 10.1167/iovs.10-7081

    Figure Lengend Snippet: Relative levels of Fas ( A ), FADD ( B ), caspase-3 ( C ), caspase-8 ( D ), and caspase-9 ( E ) mRNA transcripts evaluated by real-time PCR as well as caspase-3 ( F ), caspase-8 ( G ), and caspase-9 ( H ) activities. Data were shown in mean ± SEM.

    Article Snippet: After three washes in phosphate-buffered saline (PBS, pH 7.2), tissue samples were incubated with polyclonal rabbit anti–IFN-γ receptor (IFN-γR) (1:100, sc-700; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti–AC-caspase-3 (1:100; BD PharMingen, San Diego, CA), rabbit anti–AC-caspase-8 (1:100; Novus Biologicals, Littleton, CO), or rabbit anti–AC-caspase-9 (1:100; Novus Biologicals) primary antibodies at 4°C overnight.

    Techniques: Real-time Polymerase Chain Reaction

    Immunohistochemical and western-blot analysis of Apaf-1, active caspase-9, and active caspase-3 proteins. (A1) IHC analysis of livers in control group, (A2) IHC analysis of livers in inoculated group. The primary antibody was Apaf-1 antibody, and the positive signals were observed in the cytoplasm of hepatocyte (→); (A3) A semi-quantitative analysis of the ratio of Apaf-1 positive staining to the total field; (A4) Western-blot analysis of Apaf-1 protein in hepatocyte of control group and inoculated group gerbils; ( A5 ) A semi-quantitative analysis of relative expression of Apaf-1/β-actin. (B1) IHC analysis of livers in control group, (B2) IHC analysis of livers in inoculated group. The primary antibody was active caspase-9 antibody, and the positive signals were observed in the cytoplasm of hepatocyte (→); (B3) A semi-quantitative analysis of the ratio of active caspase-9 positive staining to the total field; (B4) Western-blot analysis of active caspase-9 protein in hepatocyte of control group and inoculated group gerbils; (B5) A semi-quantitative analysis of relative expression of active caspase-9/GAPDH. (C1) IHC analysis of livers in control group, (C2) IHC analysis of livers in inoculated group. The primary antibody was active caspase-3 antibody, and the positive signals were observed in the cytoplasm of hepatocyte (→); (C3) A semi-quantitative analysis of the ratio of active caspase-3 positive staining to the total field; (C4) Western-blot analysis of active caspase-3 protein in hepatocyte of control group and inoculated group gerbils; (C5) A semi-quantitative analysis of relative expression of active caspase-3/β-actin. ∗ p

    Journal: Frontiers in Microbiology

    Article Title: Hepatitis E Virus Induces Hepatocyte Apoptosis via Mitochondrial Pathway in Mongolian Gerbils

    doi: 10.3389/fmicb.2018.00460

    Figure Lengend Snippet: Immunohistochemical and western-blot analysis of Apaf-1, active caspase-9, and active caspase-3 proteins. (A1) IHC analysis of livers in control group, (A2) IHC analysis of livers in inoculated group. The primary antibody was Apaf-1 antibody, and the positive signals were observed in the cytoplasm of hepatocyte (→); (A3) A semi-quantitative analysis of the ratio of Apaf-1 positive staining to the total field; (A4) Western-blot analysis of Apaf-1 protein in hepatocyte of control group and inoculated group gerbils; ( A5 ) A semi-quantitative analysis of relative expression of Apaf-1/β-actin. (B1) IHC analysis of livers in control group, (B2) IHC analysis of livers in inoculated group. The primary antibody was active caspase-9 antibody, and the positive signals were observed in the cytoplasm of hepatocyte (→); (B3) A semi-quantitative analysis of the ratio of active caspase-9 positive staining to the total field; (B4) Western-blot analysis of active caspase-9 protein in hepatocyte of control group and inoculated group gerbils; (B5) A semi-quantitative analysis of relative expression of active caspase-9/GAPDH. (C1) IHC analysis of livers in control group, (C2) IHC analysis of livers in inoculated group. The primary antibody was active caspase-3 antibody, and the positive signals were observed in the cytoplasm of hepatocyte (→); (C3) A semi-quantitative analysis of the ratio of active caspase-3 positive staining to the total field; (C4) Western-blot analysis of active caspase-3 protein in hepatocyte of control group and inoculated group gerbils; (C5) A semi-quantitative analysis of relative expression of active caspase-3/β-actin. ∗ p

    Article Snippet: Primary antibodies used in this study were anti-Apaf-1 (1:200, BA2373), anti-Bax (1:300, BA0315), anti-Bcl-2 (1:200, BA0412) and anti-active caspase-3 (1:300, BA3968), anti-active caspase-9 (1:300, BA0690), all obtained from Boster, Co., Ltd., Beijing, China.

    Techniques: Immunohistochemistry, Western Blot, Staining, Expressing

    Characterization of endogenous AIP expression in the brain. a , RT-PCR was performed to detect AIP and caspase-9 mRNA in various brain regions in adult rats. The primers were designed to allow the detection of both species in the same reaction. The schematic

    Journal: The Journal of Neuroscience

    Article Title: Cloning of a Novel Apaf-1-Interacting Protein: A Potent Suppressor of Apoptosis and Ischemic Neuronal Cell Death

    doi: 10.1523/JNEUROSCI.1426-04.2004

    Figure Lengend Snippet: Characterization of endogenous AIP expression in the brain. a , RT-PCR was performed to detect AIP and caspase-9 mRNA in various brain regions in adult rats. The primers were designed to allow the detection of both species in the same reaction. The schematic

    Article Snippet: In contrast, the DEVD-cleavage activity from the purified recombinant active caspase-3 (PharMingen) or caspase-9 (MBL International, Woburn, MA) was not inhibited by either the cytosolic extracts from AIP-transfected cells or the purified AIP recombinant protein (data not shown).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

    AIP directly interacts with Apaf-1 and inhibits Apaf-1-dependent caspase-9 and caspase-3 activation. a, b , Cytochrome c -dATP-triggered (Apaf-1 dependent) caspase-9- or caspase-3-like activity was abolished in PC12 cells stably overexpressing AIP. The

    Journal: The Journal of Neuroscience

    Article Title: Cloning of a Novel Apaf-1-Interacting Protein: A Potent Suppressor of Apoptosis and Ischemic Neuronal Cell Death

    doi: 10.1523/JNEUROSCI.1426-04.2004

    Figure Lengend Snippet: AIP directly interacts with Apaf-1 and inhibits Apaf-1-dependent caspase-9 and caspase-3 activation. a, b , Cytochrome c -dATP-triggered (Apaf-1 dependent) caspase-9- or caspase-3-like activity was abolished in PC12 cells stably overexpressing AIP. The

    Article Snippet: In contrast, the DEVD-cleavage activity from the purified recombinant active caspase-3 (PharMingen) or caspase-9 (MBL International, Woburn, MA) was not inhibited by either the cytosolic extracts from AIP-transfected cells or the purified AIP recombinant protein (data not shown).

    Techniques: Activation Assay, Activity Assay, Stable Transfection

    Characterization of the anti-apoptotic effect of AIP in cell lines. a , Transfection of AIP cDNA inhibited apoptosis in human 293 cells induced by transient cDNA transfection of rat caspase-9 or Apaf-1. Cell death was analyzed at 24 hr after transfection.

    Journal: The Journal of Neuroscience

    Article Title: Cloning of a Novel Apaf-1-Interacting Protein: A Potent Suppressor of Apoptosis and Ischemic Neuronal Cell Death

    doi: 10.1523/JNEUROSCI.1426-04.2004

    Figure Lengend Snippet: Characterization of the anti-apoptotic effect of AIP in cell lines. a , Transfection of AIP cDNA inhibited apoptosis in human 293 cells induced by transient cDNA transfection of rat caspase-9 or Apaf-1. Cell death was analyzed at 24 hr after transfection.

    Article Snippet: In contrast, the DEVD-cleavage activity from the purified recombinant active caspase-3 (PharMingen) or caspase-9 (MBL International, Woburn, MA) was not inhibited by either the cytosolic extracts from AIP-transfected cells or the purified AIP recombinant protein (data not shown).

    Techniques: Transfection

    ) and various caspase-9 short variants

    Journal: The Journal of Neuroscience

    Article Title: Cloning of a Novel Apaf-1-Interacting Protein: A Potent Suppressor of Apoptosis and Ischemic Neuronal Cell Death

    doi: 10.1523/JNEUROSCI.1426-04.2004

    Figure Lengend Snippet: ) and various caspase-9 short variants

    Article Snippet: In contrast, the DEVD-cleavage activity from the purified recombinant active caspase-3 (PharMingen) or caspase-9 (MBL International, Woburn, MA) was not inhibited by either the cytosolic extracts from AIP-transfected cells or the purified AIP recombinant protein (data not shown).

    Techniques:

    Construction of an AAV vector carrying the AIP expression cassette. a , The construct contains the CMV promoter and an HA tag, which allows distinction of AIP from endogenous AIP or caspase-9. SP and pA are the SV40 mRNA splicing site and polyadenylation

    Journal: The Journal of Neuroscience

    Article Title: Cloning of a Novel Apaf-1-Interacting Protein: A Potent Suppressor of Apoptosis and Ischemic Neuronal Cell Death

    doi: 10.1523/JNEUROSCI.1426-04.2004

    Figure Lengend Snippet: Construction of an AAV vector carrying the AIP expression cassette. a , The construct contains the CMV promoter and an HA tag, which allows distinction of AIP from endogenous AIP or caspase-9. SP and pA are the SV40 mRNA splicing site and polyadenylation

    Article Snippet: In contrast, the DEVD-cleavage activity from the purified recombinant active caspase-3 (PharMingen) or caspase-9 (MBL International, Woburn, MA) was not inhibited by either the cytosolic extracts from AIP-transfected cells or the purified AIP recombinant protein (data not shown).

    Techniques: Plasmid Preparation, Expressing, Construct

    Figure 4. WTp53 interacts with the p35 fragment caspase-9. ( A ) p53 was expressed using an IVT system, and immunoprecipitation was performed in the presence of IVT-expressed MDM2 or recombinant active caspase-9. ( B ) Apoptosis was induced with dATP and rCyt- c in S100 lysates of H1299 cells stably expressing p53R175H. Antibody pairs between DO-1 (p53) and the above was added to the lysates and anti-mouse coated (donor ) and anti-rabbit (acceptor) beads were used to detect interactions between the proteins (top panel). Fluorescence signal were measured for lysates induced for apoptosis and expressed as a ratio over signal obtained for uninduced lysates for each antibody pair (bottom left panel). Immunoblots for p53 and caspase-9 was done for the lysates used for this assay (bottom right panel).

    Journal: Cell Cycle

    Article Title: Wild-type and mutant p53 mediate cisplatin resistance through interaction and inhibition of active caspase-9

    doi: 10.4161/cc.23054

    Figure Lengend Snippet: Figure 4. WTp53 interacts with the p35 fragment caspase-9. ( A ) p53 was expressed using an IVT system, and immunoprecipitation was performed in the presence of IVT-expressed MDM2 or recombinant active caspase-9. ( B ) Apoptosis was induced with dATP and rCyt- c in S100 lysates of H1299 cells stably expressing p53R175H. Antibody pairs between DO-1 (p53) and the above was added to the lysates and anti-mouse coated (donor ) and anti-rabbit (acceptor) beads were used to detect interactions between the proteins (top panel). Fluorescence signal were measured for lysates induced for apoptosis and expressed as a ratio over signal obtained for uninduced lysates for each antibody pair (bottom left panel). Immunoblots for p53 and caspase-9 was done for the lysates used for this assay (bottom right panel).

    Article Snippet: For caspase-9 activity assays, fluorescence caspase-9 substrate, Red-LEHD-FMK from Caspase-9 Detection Kit (Calbiochem; #QIA116) was added to the cells 1 h before images were taken.

    Techniques: Immunoprecipitation, Recombinant, Stable Transfection, Expressing, Fluorescence, Western Blot

    Figure 2. Different cytoplasmic levels of p53 affect cleavage of caspase-9. ( A ) Recombinant WT and mutant p53 were added to cytochrome- c challenged S100 lysates of HCT116 p53 -/- cells and the caspase cleavage profiles were probed. ( B ) Endogenous p53 in HEK293 cells was knocked-down using siRNA and the S100 cytosolic extracts of the cells were induced with recombinant cytochrome- c to initiate apoptosis. ( C ) Immunocytochemistry showing the localization of p53 R175H and p53 R273H in H1299 cells stably expressing these mutants (left panel). Immunoblots of p53 and caspase-9 of the cytochrome- c challenged S100 lysates from each cell type at different time points were probed (right panel).

    Journal: Cell Cycle

    Article Title: Wild-type and mutant p53 mediate cisplatin resistance through interaction and inhibition of active caspase-9

    doi: 10.4161/cc.23054

    Figure Lengend Snippet: Figure 2. Different cytoplasmic levels of p53 affect cleavage of caspase-9. ( A ) Recombinant WT and mutant p53 were added to cytochrome- c challenged S100 lysates of HCT116 p53 -/- cells and the caspase cleavage profiles were probed. ( B ) Endogenous p53 in HEK293 cells was knocked-down using siRNA and the S100 cytosolic extracts of the cells were induced with recombinant cytochrome- c to initiate apoptosis. ( C ) Immunocytochemistry showing the localization of p53 R175H and p53 R273H in H1299 cells stably expressing these mutants (left panel). Immunoblots of p53 and caspase-9 of the cytochrome- c challenged S100 lysates from each cell type at different time points were probed (right panel).

    Article Snippet: For caspase-9 activity assays, fluorescence caspase-9 substrate, Red-LEHD-FMK from Caspase-9 Detection Kit (Calbiochem; #QIA116) was added to the cells 1 h before images were taken.

    Techniques: Recombinant, Mutagenesis, Immunocytochemistry, Stable Transfection, Expressing, Western Blot

    Figure 3. WT and mutant p53 interacts directly with caspase-9. ( A ) One µM of recombinant p53 was incubated with recombinant active caspase-9 and the caspase-9 activity was measured after 1 h incubation (left panel). Increasing concentrations of the various recombinant p53 used in ( A ) were incubated with recombinant active caspase-9, and the caspase-9 activity was measured after 1 h incubation (right panel). ( B ) Procaspase-3 was expressed using an IVT system and the recombinant active caspase-9-induced cleavage was probed in the presence of control proteins and recombinant WTp53 after 2 h incubation. ( C ) One µM of recombinant p53 was added to recombinant active caspase-3 (left panel) or caspase-6 (right panel) and the activities of the respective caspases were measured after 1 h incubation.

    Journal: Cell Cycle

    Article Title: Wild-type and mutant p53 mediate cisplatin resistance through interaction and inhibition of active caspase-9

    doi: 10.4161/cc.23054

    Figure Lengend Snippet: Figure 3. WT and mutant p53 interacts directly with caspase-9. ( A ) One µM of recombinant p53 was incubated with recombinant active caspase-9 and the caspase-9 activity was measured after 1 h incubation (left panel). Increasing concentrations of the various recombinant p53 used in ( A ) were incubated with recombinant active caspase-9, and the caspase-9 activity was measured after 1 h incubation (right panel). ( B ) Procaspase-3 was expressed using an IVT system and the recombinant active caspase-9-induced cleavage was probed in the presence of control proteins and recombinant WTp53 after 2 h incubation. ( C ) One µM of recombinant p53 was added to recombinant active caspase-3 (left panel) or caspase-6 (right panel) and the activities of the respective caspases were measured after 1 h incubation.

    Article Snippet: For caspase-9 activity assays, fluorescence caspase-9 substrate, Red-LEHD-FMK from Caspase-9 Detection Kit (Calbiochem; #QIA116) was added to the cells 1 h before images were taken.

    Techniques: Mutagenesis, Recombinant, Incubation, Activity Assay

    Figure 5. Resistance to cisplatin in the presence of p53 correlated with the inhibition of caspase-9 activity. ( A ) H1299 cells were induced with ponasterone A to express p53 prior to treatment with 25 µM cisplatin for 48 h. After 48 h with cisplatin, caspase-9 activity was measured and normalized to cell density (top panel). The cells were then allowed to recover in the absence of cisplatin over 12 d. Caspase-9 activity after the 12-d recovery period was also measured (bottom panel). ( B ) HCT-116 p53 -/- cells were transfected with the various GFP-tagged p53 before 0.5 mM of cisplatin was added. At 72 h, substrates that are specific to the action of active caspase-9 were added to the cells to yield a fluorescent product 1 h prior to imaging. Cells positive for both GFP fluorescence and caspase-9 activity were counted and expressed as a ratio over the total number of GFP-fluorescence-positive cells (left panel). The right panel shows the representative images used to calculate the correlation percentages.

    Journal: Cell Cycle

    Article Title: Wild-type and mutant p53 mediate cisplatin resistance through interaction and inhibition of active caspase-9

    doi: 10.4161/cc.23054

    Figure Lengend Snippet: Figure 5. Resistance to cisplatin in the presence of p53 correlated with the inhibition of caspase-9 activity. ( A ) H1299 cells were induced with ponasterone A to express p53 prior to treatment with 25 µM cisplatin for 48 h. After 48 h with cisplatin, caspase-9 activity was measured and normalized to cell density (top panel). The cells were then allowed to recover in the absence of cisplatin over 12 d. Caspase-9 activity after the 12-d recovery period was also measured (bottom panel). ( B ) HCT-116 p53 -/- cells were transfected with the various GFP-tagged p53 before 0.5 mM of cisplatin was added. At 72 h, substrates that are specific to the action of active caspase-9 were added to the cells to yield a fluorescent product 1 h prior to imaging. Cells positive for both GFP fluorescence and caspase-9 activity were counted and expressed as a ratio over the total number of GFP-fluorescence-positive cells (left panel). The right panel shows the representative images used to calculate the correlation percentages.

    Article Snippet: For caspase-9 activity assays, fluorescence caspase-9 substrate, Red-LEHD-FMK from Caspase-9 Detection Kit (Calbiochem; #QIA116) was added to the cells 1 h before images were taken.

    Techniques: Inhibition, Activity Assay, Transfection, Imaging, Fluorescence

    NS3694 blocks the formation of the ∼700-kDa apoptosome complex. (A) Increasing concentrations of NS3694 were coincubated with THP.1 cell lysates (10 mg) in the presence (+) and absence (−) of 2 mM dATP-MgCl 2 for 30 min at 37°C before assaying for DEVDase activity (essentially caspases 3 and 7) and caspase 9 (Casp-9) and caspase 3 (Casp-3) processing as described in Materials and Methods. The positions of the procaspases (Pro) and processed forms (p35 and p37 [p35/37] and p19 and p17 [p19/p17]) are indicated to the right of the blots. NS3694 was added in a dimethyl sulfoxide solution, and control samples were incubated with equivalent amounts of the solvent, which did not inhibit caspase activation. (B) Two-milligram aliquots of control THP.1 cell lysates and dATP-activated THP.1 cell lysates in the presence and absence of NS3694 (500 μM) were fractionated on Superose 6 columns, and the fractions were analyzed for Apaf-1 and caspase 9 (Casp-9). The sizes of the various peaks were determined by using gel filtration marker proteins, and the elution positions of the ∼700-kDa and ∼1.4-MDa apoptosome complexes and aldolase (158 kDa) are indicated above the blots. Processed caspase 9 was not detected in the control samples and Ns3694-treated samples. In the dATP-activated sample, procaspase 9 (pro) was totally processed to its active subunits. The results shown are from a representative experiment, and similar data were obtained in a repeated experiment. Numbers in boxes, fraction numbers.

    Journal: Molecular and Cellular Biology

    Article Title: Diarylurea Compounds Inhibit Caspase Activation by Preventing the Formation of the Active 700-Kilodalton Apoptosome Complex

    doi: 10.1128/MCB.23.21.7829-7837.2003

    Figure Lengend Snippet: NS3694 blocks the formation of the ∼700-kDa apoptosome complex. (A) Increasing concentrations of NS3694 were coincubated with THP.1 cell lysates (10 mg) in the presence (+) and absence (−) of 2 mM dATP-MgCl 2 for 30 min at 37°C before assaying for DEVDase activity (essentially caspases 3 and 7) and caspase 9 (Casp-9) and caspase 3 (Casp-3) processing as described in Materials and Methods. The positions of the procaspases (Pro) and processed forms (p35 and p37 [p35/37] and p19 and p17 [p19/p17]) are indicated to the right of the blots. NS3694 was added in a dimethyl sulfoxide solution, and control samples were incubated with equivalent amounts of the solvent, which did not inhibit caspase activation. (B) Two-milligram aliquots of control THP.1 cell lysates and dATP-activated THP.1 cell lysates in the presence and absence of NS3694 (500 μM) were fractionated on Superose 6 columns, and the fractions were analyzed for Apaf-1 and caspase 9 (Casp-9). The sizes of the various peaks were determined by using gel filtration marker proteins, and the elution positions of the ∼700-kDa and ∼1.4-MDa apoptosome complexes and aldolase (158 kDa) are indicated above the blots. Processed caspase 9 was not detected in the control samples and Ns3694-treated samples. In the dATP-activated sample, procaspase 9 (pro) was totally processed to its active subunits. The results shown are from a representative experiment, and similar data were obtained in a repeated experiment. Numbers in boxes, fraction numbers.

    Article Snippet: Fractions (0.5 ml) were collected and analyzed by Western blotting for Apaf-1 and caspase 9.

    Techniques: Activity Assay, Incubation, Activation Assay, Filtration, Marker, Multiple Displacement Amplification

    Diarylurea compounds do not inhibit the activity of caspase 9 or caspase 3. The enzymatic activity of recombinant caspase 9 (rCaspase-9) (A) and recombinant caspase 3 (rCaspase-3) (B) was assessed by spectrofluorometric quantification of LEHD-AFC or DEVD-AFC cleavage, respectively, in the presence (+) or absence of 100 μM NS3694, NS1784, NS1764, DEVD-CHO (DEVD), or zVAD-fmk (zVAD). Enzymatic activity is presented as picomoles per minute, and the means ± standard deviations (error bars) for three samples are shown. Data are representative of the results from three independent assays.

    Journal: Molecular and Cellular Biology

    Article Title: Diarylurea Compounds Inhibit Caspase Activation by Preventing the Formation of the Active 700-Kilodalton Apoptosome Complex

    doi: 10.1128/MCB.23.21.7829-7837.2003

    Figure Lengend Snippet: Diarylurea compounds do not inhibit the activity of caspase 9 or caspase 3. The enzymatic activity of recombinant caspase 9 (rCaspase-9) (A) and recombinant caspase 3 (rCaspase-3) (B) was assessed by spectrofluorometric quantification of LEHD-AFC or DEVD-AFC cleavage, respectively, in the presence (+) or absence of 100 μM NS3694, NS1784, NS1764, DEVD-CHO (DEVD), or zVAD-fmk (zVAD). Enzymatic activity is presented as picomoles per minute, and the means ± standard deviations (error bars) for three samples are shown. Data are representative of the results from three independent assays.

    Article Snippet: Fractions (0.5 ml) were collected and analyzed by Western blotting for Apaf-1 and caspase 9.

    Techniques: Activity Assay, Recombinant

    NS3694 inhibits the association of caspase 9 to Apaf-1. Cytosolic extracts prepared from HeLa cells were left untreated (UN) or incubated with cytochrome c (Cc; 1 μM) and dATP (1 mM), in the presence (+) or absence of 100 μM NS3694 or 2 μM zVAD-fmk (zVAD) at 37°C. After 1-h incubation, caspase 9 was immunoprecipitated with anti-caspase 9 antibody precoupled to Sepharose beads. The supernatants (Sup) and immunoprecipitates (IP) were separated by SDS-PAGE followed by immunoblot analysis using antibodies against the indicated proteins. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a control for the specificity of the immunoprecipitation and was detected only in the supernatants. Bands corresponding to the heavy chain (IgG) of the immunoprecipitating antibody are also visible. The experiment was repeated once with essentially identical results. WB, Western blotting.

    Journal: Molecular and Cellular Biology

    Article Title: Diarylurea Compounds Inhibit Caspase Activation by Preventing the Formation of the Active 700-Kilodalton Apoptosome Complex

    doi: 10.1128/MCB.23.21.7829-7837.2003

    Figure Lengend Snippet: NS3694 inhibits the association of caspase 9 to Apaf-1. Cytosolic extracts prepared from HeLa cells were left untreated (UN) or incubated with cytochrome c (Cc; 1 μM) and dATP (1 mM), in the presence (+) or absence of 100 μM NS3694 or 2 μM zVAD-fmk (zVAD) at 37°C. After 1-h incubation, caspase 9 was immunoprecipitated with anti-caspase 9 antibody precoupled to Sepharose beads. The supernatants (Sup) and immunoprecipitates (IP) were separated by SDS-PAGE followed by immunoblot analysis using antibodies against the indicated proteins. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a control for the specificity of the immunoprecipitation and was detected only in the supernatants. Bands corresponding to the heavy chain (IgG) of the immunoprecipitating antibody are also visible. The experiment was repeated once with essentially identical results. WB, Western blotting.

    Article Snippet: Fractions (0.5 ml) were collected and analyzed by Western blotting for Apaf-1 and caspase 9.

    Techniques: Incubation, Immunoprecipitation, SDS Page, Western Blot

    A utophagy-deficient mice showed increased activity of apoptosis and necroptosis. ( a ) Depletion of Atg7 increased pancreatic caspase-3 in 20-week-old Atg7 Δpan mice. Caspase-3 quantitation and representative IF microphotographs of Atg7 F/F ( n =4) and Atg7 Δpan ( n =4) pancreatic tissue stained for DAPI (blue) and Caspase-3 (red) (anti-active caspase-3 ab2302, 1/50, scale bar=50 μ m). ( b ) Reduced pancreatic Atg7 level increased the expression of caspase-8 in 12-week-old Atg7 Δpan mice. Caspase-8 quantitation and representative IF microphotographs of Atg7 F/F ( n =5) and Atg7 Δpan ( n =5) pancreatic tissue stained for DAPI (blue) and Caspase-8 (green) (anti-active caspase-8 Novusbio NB100-56116, 1/1000, scale bar=50 μ m). ( c ) Increased the expression of caspase-9 in 12-week-old Atg7 Δpan mice. Caspase-9 quantitation and representative IF microphotographs of Atg7 F/F ( n =5) and Atg7 Δpan ( n =5) pancreatic tissue stained for DAPI (blue) and Caspase-9 (green) (anti-caspase-9 Novusbio NB100-56118, 1/1000, scale bar=50 μ m). ( d ) Reduced pancreatic Atg7 level increased the expression of Bax in 12-week-old Atg7 Δpan mice Bax quantitation and representative IF microphotographs of Atg7 F/F ( n =5) and Atg7 Δpan ( n =5) pancreatic tissue stained for DAPI (blue) and Bax (green) (anti-Bax sc-526, 1/50, scale bar=50 μ m). ( e ) Reduced pancreatic Atg7 level increased the expression of necroptotic protein Rip3 in 20-week-old Atg7 Δpan mice. Rip3 quantitation and representative IF microphotographs of Atg7 F/F ( n =5) and Atg7 Δpan ( n =5) pancreatic tissue stained for DAPI (blue) and Rip3 (green) (anti-Rip3 ab62344, 1/200, scale bar=50 μ m). ( f ) Reduced pancreatic Atg7 level increased the expression of necroptotic protein Mlkl in 20-week-old Atg7 Δpan mice. Mlkl quantitation and representative IF microphotographs of Atg7 F/F ( n =5) and Atg7 Δpan ( n =5) pancreatic tissue stained for DAPI (blue) and Mlkl (green) (anti-Mlkl ab194699, 1/100, scale bar=50 μ m). ( g ) Reduced pancreatic Atg7 level increased the expression of necroptotic protein Hmgb1 in 20-week-old Atg7 Δpan mice. Hmgb1 quantitation and representative IF microphotographs of Atg7 F/F ( n =5) and Atg7 Δpan ( n =5) pancreatic tissue stained for DAPI (blue) and Hmgb1 (red) (anti-Hmgb1 ab18256, 1/1000, scale bar=50 μ m). Data are mean±S.E.M. for the numbers of animals as indicated in the graph, * P

    Journal: Cell Death & Disease

    Article Title: RIP3 attenuates the pancreatic damage induced by deletion of ATG7

    doi: 10.1038/cddis.2017.313

    Figure Lengend Snippet: A utophagy-deficient mice showed increased activity of apoptosis and necroptosis. ( a ) Depletion of Atg7 increased pancreatic caspase-3 in 20-week-old Atg7 Δpan mice. Caspase-3 quantitation and representative IF microphotographs of Atg7 F/F ( n =4) and Atg7 Δpan ( n =4) pancreatic tissue stained for DAPI (blue) and Caspase-3 (red) (anti-active caspase-3 ab2302, 1/50, scale bar=50 μ m). ( b ) Reduced pancreatic Atg7 level increased the expression of caspase-8 in 12-week-old Atg7 Δpan mice. Caspase-8 quantitation and representative IF microphotographs of Atg7 F/F ( n =5) and Atg7 Δpan ( n =5) pancreatic tissue stained for DAPI (blue) and Caspase-8 (green) (anti-active caspase-8 Novusbio NB100-56116, 1/1000, scale bar=50 μ m). ( c ) Increased the expression of caspase-9 in 12-week-old Atg7 Δpan mice. Caspase-9 quantitation and representative IF microphotographs of Atg7 F/F ( n =5) and Atg7 Δpan ( n =5) pancreatic tissue stained for DAPI (blue) and Caspase-9 (green) (anti-caspase-9 Novusbio NB100-56118, 1/1000, scale bar=50 μ m). ( d ) Reduced pancreatic Atg7 level increased the expression of Bax in 12-week-old Atg7 Δpan mice Bax quantitation and representative IF microphotographs of Atg7 F/F ( n =5) and Atg7 Δpan ( n =5) pancreatic tissue stained for DAPI (blue) and Bax (green) (anti-Bax sc-526, 1/50, scale bar=50 μ m). ( e ) Reduced pancreatic Atg7 level increased the expression of necroptotic protein Rip3 in 20-week-old Atg7 Δpan mice. Rip3 quantitation and representative IF microphotographs of Atg7 F/F ( n =5) and Atg7 Δpan ( n =5) pancreatic tissue stained for DAPI (blue) and Rip3 (green) (anti-Rip3 ab62344, 1/200, scale bar=50 μ m). ( f ) Reduced pancreatic Atg7 level increased the expression of necroptotic protein Mlkl in 20-week-old Atg7 Δpan mice. Mlkl quantitation and representative IF microphotographs of Atg7 F/F ( n =5) and Atg7 Δpan ( n =5) pancreatic tissue stained for DAPI (blue) and Mlkl (green) (anti-Mlkl ab194699, 1/100, scale bar=50 μ m). ( g ) Reduced pancreatic Atg7 level increased the expression of necroptotic protein Hmgb1 in 20-week-old Atg7 Δpan mice. Hmgb1 quantitation and representative IF microphotographs of Atg7 F/F ( n =5) and Atg7 Δpan ( n =5) pancreatic tissue stained for DAPI (blue) and Hmgb1 (red) (anti-Hmgb1 ab18256, 1/1000, scale bar=50 μ m). Data are mean±S.E.M. for the numbers of animals as indicated in the graph, * P

    Article Snippet: F4/80 (NBP2-12506), Active/cleaved Caspase-8 (NB100-56116), active/cleaved Caspase-9 (NB100-56118) was obtained from Novus Biologicals (Cambridge, UK).

    Techniques: Mouse Assay, Activity Assay, Quantitation Assay, Staining, Expressing

    Exacerbated apoptosis and altered inflammation in double-deficient Atg7 Δpan -Rip3 −/− mice. ( a ) Depletion of Rip3 and pancreatic Atg7 (Atg7 Δpan -Rip3 d/d ) increased pancreatic caspase-3 in 12-week-old mice. Caspase-3 quantitation and representative IF microphotographs of Atg7 Δpan ( n =5) and Atg7 Δpan -Rip3 d/d ( n =5) pancreatic tissue stained for DAPI (blue) and Caspase-3 (red) (anti-active caspase-3 ab2302, 1/50, scale bar=50 μ m). ( b ) Depletion of Rip3 and pancreatic Atg7 (Atg7 Δpan -Rip3 d/d ) increased pancreatic Bax in 12-week-old mice. Bax quantitation and representative IF microphotographs of Atg7 Δpan ( n =5) and Atg7 Δpan -Rip3 d/d ( n =5) pancreatic tissue stained for DAPI (blue) and Caspase-3 (red) (anti-Bax (sc-526, 1/50). ( c ) Depletion of Rip3 and pancreatic Atg7 (Atg7 Δpan -Rip3 d/d ) increased pancreatic caspase-9 in 12-week-old mice. Caspase-9 quantitation and representative IF microphotographs of Atg7 Δpan ( n =5) and Atg7 Δpan -Rip3 d/d ( n =5) pancreatic tissue stained for DAPI (blue) and Caspase-9 (red) (anti-caspase-9 NB100-56118, 1/1000). ( d ) Reduced infiltration inflammation of macrophages (F4/80, NBP2-12506, 1/75) and early T-lymphocytes (MPO, ab9535, 1/50) in double-deficient Atg7 Δpan -Rip3 d/d ( n =10) mice compared with pancreatic Atg7 Δpan ( n =7). Data are mean±S.E.M. for the numbers of animals as indicated in the graph, * P

    Journal: Cell Death & Disease

    Article Title: RIP3 attenuates the pancreatic damage induced by deletion of ATG7

    doi: 10.1038/cddis.2017.313

    Figure Lengend Snippet: Exacerbated apoptosis and altered inflammation in double-deficient Atg7 Δpan -Rip3 −/− mice. ( a ) Depletion of Rip3 and pancreatic Atg7 (Atg7 Δpan -Rip3 d/d ) increased pancreatic caspase-3 in 12-week-old mice. Caspase-3 quantitation and representative IF microphotographs of Atg7 Δpan ( n =5) and Atg7 Δpan -Rip3 d/d ( n =5) pancreatic tissue stained for DAPI (blue) and Caspase-3 (red) (anti-active caspase-3 ab2302, 1/50, scale bar=50 μ m). ( b ) Depletion of Rip3 and pancreatic Atg7 (Atg7 Δpan -Rip3 d/d ) increased pancreatic Bax in 12-week-old mice. Bax quantitation and representative IF microphotographs of Atg7 Δpan ( n =5) and Atg7 Δpan -Rip3 d/d ( n =5) pancreatic tissue stained for DAPI (blue) and Caspase-3 (red) (anti-Bax (sc-526, 1/50). ( c ) Depletion of Rip3 and pancreatic Atg7 (Atg7 Δpan -Rip3 d/d ) increased pancreatic caspase-9 in 12-week-old mice. Caspase-9 quantitation and representative IF microphotographs of Atg7 Δpan ( n =5) and Atg7 Δpan -Rip3 d/d ( n =5) pancreatic tissue stained for DAPI (blue) and Caspase-9 (red) (anti-caspase-9 NB100-56118, 1/1000). ( d ) Reduced infiltration inflammation of macrophages (F4/80, NBP2-12506, 1/75) and early T-lymphocytes (MPO, ab9535, 1/50) in double-deficient Atg7 Δpan -Rip3 d/d ( n =10) mice compared with pancreatic Atg7 Δpan ( n =7). Data are mean±S.E.M. for the numbers of animals as indicated in the graph, * P

    Article Snippet: F4/80 (NBP2-12506), Active/cleaved Caspase-8 (NB100-56116), active/cleaved Caspase-9 (NB100-56118) was obtained from Novus Biologicals (Cambridge, UK).

    Techniques: Mouse Assay, Quantitation Assay, Staining

    Altered expression of Pitx2, Tbx5 , and Myocd in response to atrial short-term tachyarrhythmia. (a) Average relative values of transcript levels of Pitx2c , Tbx5 , and Myocd in the left atrium from paced (EXP) versus sham (CON) animals. ∗ p ≤ 0.05 ( n = 5 for each group). (b) Representative Western blots of left atrium samples from sham-operated (CON, lines 1-2) and paced (EXP, lines 3 and 4) animals and overall relative levels of the proteins as based on average values from each group. ∗ p ≤ 0.05 ( n = 5 for each group). Western blot replicates were probed with antibodies against PITX2A,B,C, TBX5, MYOCD (myocardin), CASP3 (caspase-3), CASP9 (caspase-9), SERCA2 (cardiac sarcoplasmic reticulum Ca(2+)-ATPase 2a), CASQ2 (cardiac calsequestrin), TNNI3 (cardiac troponin I), and GAPDH (glyceraldehyde-3-phosphate dehydrogenase). MW values of the bands detected are shown.

    Journal: BioMed Research International

    Article Title: A MicroRNA-Transcription Factor Blueprint for Early Atrial Arrhythmogenic Remodeling

    doi: 10.1155/2015/263151

    Figure Lengend Snippet: Altered expression of Pitx2, Tbx5 , and Myocd in response to atrial short-term tachyarrhythmia. (a) Average relative values of transcript levels of Pitx2c , Tbx5 , and Myocd in the left atrium from paced (EXP) versus sham (CON) animals. ∗ p ≤ 0.05 ( n = 5 for each group). (b) Representative Western blots of left atrium samples from sham-operated (CON, lines 1-2) and paced (EXP, lines 3 and 4) animals and overall relative levels of the proteins as based on average values from each group. ∗ p ≤ 0.05 ( n = 5 for each group). Western blot replicates were probed with antibodies against PITX2A,B,C, TBX5, MYOCD (myocardin), CASP3 (caspase-3), CASP9 (caspase-9), SERCA2 (cardiac sarcoplasmic reticulum Ca(2+)-ATPase 2a), CASQ2 (cardiac calsequestrin), TNNI3 (cardiac troponin I), and GAPDH (glyceraldehyde-3-phosphate dehydrogenase). MW values of the bands detected are shown.

    Article Snippet: Antibodies The following primary antibodies were used: (1) rabbit polyclonal antibodies to PITX2A,B,C (Capra Science, Ängelholm, Sweden; at 1 : 2000 dilution): the specificity of the anti-PITX2 antibodies was independently validated by Western blot analysis of COS-7 cells expressing each PITX2 isoform [ ]; (2) rabbit polyclonal antibodies to TBX5 (Abcam, Cambridge, UK; at 1 : 500 dilution); (3) rabbit polyclonal antibodies to myocardin which were generated by Davids Biotechnologie (Regensburg, Germany) using the recombinant TAD-containing fragment of porcine MYOCD as immunogen (at 1 : 500 dilution): these antibodies were shown to be specific for both MYOCD-A (minor) and MYOCD-B (major) variants expressed in pig cardiac tissues [ ]; (4) rabbit polyclonal antibodies to cardiac troponin I (Abcam, Cambridge, UK; at 1 : 40000 dilution); (5) rabbit polyclonal antibodies to cardiac calsequestrin-2 (Abcam, Cambridge, UK; at 1 : 10000 dilution); (6) rabbit monoclonal antibodies to sarcoplasmic reticulum ATPase (SERCA-2A; Abcam, Cambridge, UK; at 1 : 10000 dilution); (7) rabbit polyclonal antibodies to caspase-3 (Cell Signaling, Leiden, Netherlands; at 1 : 1000); (8) rabbit monoclonal antibodies to caspase-9 (Abcam, Cambridge, UK; at 1 : 1000 dilution); and (9) mouse monoclonal anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) antibody (Sigma, Madrid, Spain; at 1 : 10000 dilution).

    Techniques: Expressing, Western Blot

    AAV-mediated TRAIL overexpression induced increased expression of caspase-3, caspase-8, and caspase-9, and reduced expression of Bcl-2 in OSCC cells. (A) Immunofluorescence assay was carried out to detect the infection efficiency of the recombinant AAV system in the SCC25 OSCC cell line (magnification, ×400). The left panel presents light microscopy and the right field presents fluorescence microscopy. (B) Reverse transcription-quantitative polymerase chain reaction, (C) western blotting and (D) immunocytochemistry analysis (magnification, ×200) were performed to measure apoptosis-related genes expression changes in SCC25 cells with upregulation of TRAIL. Data are presented as the mean ± standard deviation. Each assay was performed independently at least three times. # P

    Journal: International Journal of Molecular Medicine

    Article Title: Telomerase reverse transcriptase interference synergistically promotes tumor necrosis factor-related apoptosis-inducing ligand-induced oral squamous cell carcinoma apoptosis and suppresses proliferation in vitro and in vivo

    doi: 10.3892/ijmm.2018.3721

    Figure Lengend Snippet: AAV-mediated TRAIL overexpression induced increased expression of caspase-3, caspase-8, and caspase-9, and reduced expression of Bcl-2 in OSCC cells. (A) Immunofluorescence assay was carried out to detect the infection efficiency of the recombinant AAV system in the SCC25 OSCC cell line (magnification, ×400). The left panel presents light microscopy and the right field presents fluorescence microscopy. (B) Reverse transcription-quantitative polymerase chain reaction, (C) western blotting and (D) immunocytochemistry analysis (magnification, ×200) were performed to measure apoptosis-related genes expression changes in SCC25 cells with upregulation of TRAIL. Data are presented as the mean ± standard deviation. Each assay was performed independently at least three times. # P

    Article Snippet: In the present study, the expression of TRAIL triggered the apoptosis process and pathway, including the upregulation of caspase-8, caspase-9 and caspase-3, as well as the inhibition of Bcl-2.

    Techniques: Over Expression, Expressing, Immunofluorescence, Infection, Recombinant, Light Microscopy, Fluorescence, Microscopy, Real-time Polymerase Chain Reaction, Western Blot, Immunocytochemistry, Standard Deviation

    Expression of, and proteolytic activity assay for, recombinant caspase-3/9. ( a ) SDS-PAGE analysis of recombinant procaspase-3/9-His 6 expressed in E . coli . ( b ) Specific proteolytic activity of recombinant caspase-3/9-His 6 . Cell lysate from E . coli either transformed with a vector encoding caspase-3/9-His 6 (casp) or control vector (vec) were analyzed for caspase-3 (DEVD), -8 (IETD), and -9 (LEHD) catalytic activity using Ac-DEVD-MCA, Ac-IETD-MCA and Ac-LEHD-MCA, respectively. ( c ) DEVDase activity of recombinant caspase-3/9-His 6 expressed at different temperatures. Cell lysate from E . coli without IPTG induction, with IPTG induction at 37 °C, and with IPTG induction at 15 °C were analyzed for DEVDase activity using Ac-DEVD-MCA. ( d ) Microinjection of caspase-3/9-His 6 into oocytes. Purified caspase-3/9-His 6 (1.1 µ g/mL at a final concentration) or control buffer was microinjected into immature oocytes, and photographs were taken at the indicated times after microinjection. ( e ) The number of apoptotic eggs was counted after microinjection of purified caspase-3/9-His 6 (closed triangle, 1.1 µ g/mL; open triangle, 0.56 µ g/mL at a final concentration) or control buffer (circle). The results are representative of four independent experiments.

    Journal: Scientific Reports

    Article Title: Starfish Apaf-1 activates effector caspase-3/9 upon apoptosis of aged eggs

    doi: 10.1038/s41598-018-19845-6

    Figure Lengend Snippet: Expression of, and proteolytic activity assay for, recombinant caspase-3/9. ( a ) SDS-PAGE analysis of recombinant procaspase-3/9-His 6 expressed in E . coli . ( b ) Specific proteolytic activity of recombinant caspase-3/9-His 6 . Cell lysate from E . coli either transformed with a vector encoding caspase-3/9-His 6 (casp) or control vector (vec) were analyzed for caspase-3 (DEVD), -8 (IETD), and -9 (LEHD) catalytic activity using Ac-DEVD-MCA, Ac-IETD-MCA and Ac-LEHD-MCA, respectively. ( c ) DEVDase activity of recombinant caspase-3/9-His 6 expressed at different temperatures. Cell lysate from E . coli without IPTG induction, with IPTG induction at 37 °C, and with IPTG induction at 15 °C were analyzed for DEVDase activity using Ac-DEVD-MCA. ( d ) Microinjection of caspase-3/9-His 6 into oocytes. Purified caspase-3/9-His 6 (1.1 µ g/mL at a final concentration) or control buffer was microinjected into immature oocytes, and photographs were taken at the indicated times after microinjection. ( e ) The number of apoptotic eggs was counted after microinjection of purified caspase-3/9-His 6 (closed triangle, 1.1 µ g/mL; open triangle, 0.56 µ g/mL at a final concentration) or control buffer (circle). The results are representative of four independent experiments.

    Article Snippet: Each membrane was blocked with PBS–T (0.05% Tween20-PBS) containing 1% BSA (Sigma-Aldrich), and was incubated with the anti-caspase-3/9 antibody, anti- ERK1/2 antibody (CST), and anti-active p38MAPK antibody (Promega) at a dilution of 1:2000 for 1 h at room temperature.

    Techniques: Expressing, Activity Assay, Recombinant, SDS Page, Transformation Assay, Plasmid Preparation, Purification, Concentration Assay

    Apoptosome-like complex formation by recombinant procaspase-3/9-His 6 in the cell-free preparations. ( a ) Western blot analysis of recombinant GST-sfApaf-1 CARD (1–134 aa) protein using anti-sfApaf-1 antibody. Lanes: (1) without IPTG induction; (2) with IPTG induction at 37 °C. ( b ) Ultracentrifuged cell-free preparations were fractionated by gel filtration chromatography. Endogenous procaspase-3/9 and sfApaf-1 were detected by western blotting with anti-caspase-3/9 and anti-sfApaf-1 antibodies. ( c ) Activation of endogenous caspase-3/9 in cell-free preparations by treatment with procaspase-3/9-His 6 . A time course of DEVDase activity was measured at the indicated times after adding either procaspase-3/9-His 6 or buffer (control). ( d ) Ultracentrifuged cell-free preparations were incubated with recombinant procaspase-3/9-His 6 , and fractionated by gel filtration chromatography. Fractions were analyzed by western blotting with the anti-caspase-3/9 antibody and anti-sfApaf-1 antibody. ( e ) DEVDase activity in fractions was measured by the cleavage of Ac-DEVD-MCA. The red column is from gel filtered cell-free preparations with procaspase-3/9-His 6 . The results are representative of two independent experiments.

    Journal: Scientific Reports

    Article Title: Starfish Apaf-1 activates effector caspase-3/9 upon apoptosis of aged eggs

    doi: 10.1038/s41598-018-19845-6

    Figure Lengend Snippet: Apoptosome-like complex formation by recombinant procaspase-3/9-His 6 in the cell-free preparations. ( a ) Western blot analysis of recombinant GST-sfApaf-1 CARD (1–134 aa) protein using anti-sfApaf-1 antibody. Lanes: (1) without IPTG induction; (2) with IPTG induction at 37 °C. ( b ) Ultracentrifuged cell-free preparations were fractionated by gel filtration chromatography. Endogenous procaspase-3/9 and sfApaf-1 were detected by western blotting with anti-caspase-3/9 and anti-sfApaf-1 antibodies. ( c ) Activation of endogenous caspase-3/9 in cell-free preparations by treatment with procaspase-3/9-His 6 . A time course of DEVDase activity was measured at the indicated times after adding either procaspase-3/9-His 6 or buffer (control). ( d ) Ultracentrifuged cell-free preparations were incubated with recombinant procaspase-3/9-His 6 , and fractionated by gel filtration chromatography. Fractions were analyzed by western blotting with the anti-caspase-3/9 antibody and anti-sfApaf-1 antibody. ( e ) DEVDase activity in fractions was measured by the cleavage of Ac-DEVD-MCA. The red column is from gel filtered cell-free preparations with procaspase-3/9-His 6 . The results are representative of two independent experiments.

    Article Snippet: Each membrane was blocked with PBS–T (0.05% Tween20-PBS) containing 1% BSA (Sigma-Aldrich), and was incubated with the anti-caspase-3/9 antibody, anti- ERK1/2 antibody (CST), and anti-active p38MAPK antibody (Promega) at a dilution of 1:2000 for 1 h at room temperature.

    Techniques: Recombinant, Western Blot, Filtration, Chromatography, Activation Assay, Activity Assay, Incubation

    Activation and cleavage of endogenous caspase-3/9 upon apoptosis in unfertilized eggs. ( a ) CBB gel staining and western blotting analysis of recombinant caspase-3/9-His 6 . Cell lysate of E . coli expressing recombinant caspase-3/9-His 6 was subjected to SDS-PAGE, followed by CBB gel staining (left panel), or analyzed by western blotting using the anti-caspase-3/9 antibody (right panel). Lanes: (1) with induction of IPTG at 37 °C; (2) at 15 °C. ( b ) Time course of endogenous caspase-3/9 activation after 1-MA treatment. Samples of oocytes were analyzed by SDS-PAGE and western blotting with the anti- caspase-3/9 antibody. Cleaved caspase-3/9 was visible after longer exposures. At the same time, the activity of endogenous caspase-3/9 was measured by the cleavage of Ac-DEVD-MCA. The morphological changes of the oocytes/eggs were observed with a light microscope equipped with Nomarski differential interference contrast optics; (0:00) immature oocyte; (0:20–4:00) mature eggs; (8:20) blebbing egg; (9:30–11:00) fragmented eggs. ( c . The results are representative of three independent experiments.

    Journal: Scientific Reports

    Article Title: Starfish Apaf-1 activates effector caspase-3/9 upon apoptosis of aged eggs

    doi: 10.1038/s41598-018-19845-6

    Figure Lengend Snippet: Activation and cleavage of endogenous caspase-3/9 upon apoptosis in unfertilized eggs. ( a ) CBB gel staining and western blotting analysis of recombinant caspase-3/9-His 6 . Cell lysate of E . coli expressing recombinant caspase-3/9-His 6 was subjected to SDS-PAGE, followed by CBB gel staining (left panel), or analyzed by western blotting using the anti-caspase-3/9 antibody (right panel). Lanes: (1) with induction of IPTG at 37 °C; (2) at 15 °C. ( b ) Time course of endogenous caspase-3/9 activation after 1-MA treatment. Samples of oocytes were analyzed by SDS-PAGE and western blotting with the anti- caspase-3/9 antibody. Cleaved caspase-3/9 was visible after longer exposures. At the same time, the activity of endogenous caspase-3/9 was measured by the cleavage of Ac-DEVD-MCA. The morphological changes of the oocytes/eggs were observed with a light microscope equipped with Nomarski differential interference contrast optics; (0:00) immature oocyte; (0:20–4:00) mature eggs; (8:20) blebbing egg; (9:30–11:00) fragmented eggs. ( c . The results are representative of three independent experiments.

    Article Snippet: Each membrane was blocked with PBS–T (0.05% Tween20-PBS) containing 1% BSA (Sigma-Aldrich), and was incubated with the anti-caspase-3/9 antibody, anti- ERK1/2 antibody (CST), and anti-active p38MAPK antibody (Promega) at a dilution of 1:2000 for 1 h at room temperature.

    Techniques: Activation Assay, Staining, Western Blot, Recombinant, Expressing, SDS Page, Activity Assay, Light Microscopy

    Interaction of sfApaf-1 CARD with caspase-3/9 CARD. ( a ) Glutathione Sepharose 4B bead-bound GST-A-CARD was incubated with His-C-CARD in PBS buffer; glutathione Sepharose 4B-bound GST was used as a control. Associated His-C-CARD was co-eluted with GST-A-CARD (right panel), but not with GST (left panel) after the addition of elution buffer, as determined by SDS–PAGE and CBB gel staining. ( b . The results are representative of two independent experiments.

    Journal: Scientific Reports

    Article Title: Starfish Apaf-1 activates effector caspase-3/9 upon apoptosis of aged eggs

    doi: 10.1038/s41598-018-19845-6

    Figure Lengend Snippet: Interaction of sfApaf-1 CARD with caspase-3/9 CARD. ( a ) Glutathione Sepharose 4B bead-bound GST-A-CARD was incubated with His-C-CARD in PBS buffer; glutathione Sepharose 4B-bound GST was used as a control. Associated His-C-CARD was co-eluted with GST-A-CARD (right panel), but not with GST (left panel) after the addition of elution buffer, as determined by SDS–PAGE and CBB gel staining. ( b . The results are representative of two independent experiments.

    Article Snippet: Each membrane was blocked with PBS–T (0.05% Tween20-PBS) containing 1% BSA (Sigma-Aldrich), and was incubated with the anti-caspase-3/9 antibody, anti- ERK1/2 antibody (CST), and anti-active p38MAPK antibody (Promega) at a dilution of 1:2000 for 1 h at room temperature.

    Techniques: Incubation, SDS Page, Staining

    Activation of caspase-3/9 by sfApaf-1 CARD. ( a ) Activation of endogenous caspase-3/9 in cell-free preparation by GST-A-CARD. A time course of DEVDase activity was measured at the indicated time after addition of GST-A-CARD or control GST. ( b ) Cleavage of endogenous caspase-3/9 in cell-free preparations was induced by GST-A-CARD but not by GST, and detected by western blotting with the anti-caspase-3/9 antibody. ( c ) The interaction between A-CARD and caspase-3/9 in cell-free preparations. The cell-free preparation was incubated with GST-A-CARD or not, and fractionated by using gel filtration chromatography. Each fraction was analyzed by western blotting with anti-caspase-3/9 and anti-GST antibodies. ( d . The results are representative of three independent experiments.

    Journal: Scientific Reports

    Article Title: Starfish Apaf-1 activates effector caspase-3/9 upon apoptosis of aged eggs

    doi: 10.1038/s41598-018-19845-6

    Figure Lengend Snippet: Activation of caspase-3/9 by sfApaf-1 CARD. ( a ) Activation of endogenous caspase-3/9 in cell-free preparation by GST-A-CARD. A time course of DEVDase activity was measured at the indicated time after addition of GST-A-CARD or control GST. ( b ) Cleavage of endogenous caspase-3/9 in cell-free preparations was induced by GST-A-CARD but not by GST, and detected by western blotting with the anti-caspase-3/9 antibody. ( c ) The interaction between A-CARD and caspase-3/9 in cell-free preparations. The cell-free preparation was incubated with GST-A-CARD or not, and fractionated by using gel filtration chromatography. Each fraction was analyzed by western blotting with anti-caspase-3/9 and anti-GST antibodies. ( d . The results are representative of three independent experiments.

    Article Snippet: Each membrane was blocked with PBS–T (0.05% Tween20-PBS) containing 1% BSA (Sigma-Aldrich), and was incubated with the anti-caspase-3/9 antibody, anti- ERK1/2 antibody (CST), and anti-active p38MAPK antibody (Promega) at a dilution of 1:2000 for 1 h at room temperature.

    Techniques: Activation Assay, Activity Assay, Western Blot, Incubation, Filtration, Chromatography

    Both the extrinsic and intrinsic pathways are involved in Runx3-promoted cell apoptosis by dsRNA poly(I:C) and IAV infection. ( a ) Runx3 is required for the activation of caspase-8 and caspsas-9 by poly(I:C). BEAS-2B cells were transfected with 20 nM control siRNA (Con.), Runx3 siRNA-1 (Runx3-S1), or Runx3 siRNA-2 (Runx3-S2), grown for 72 h, then treated with high molecular weight poly(I:C) (2 μg/ml) for 0, 2 or 4 h. Equal amounts of cell lysates were subjected to Western blotting with specific antibodies against cleaved caspase-8, cleaved caspase-9, Runx3 or action as indicated. ( b,c ) BEAS-2B cells were infected with recombinant adenovirus containing vector alone or Runx3 and grown for 60 h. The cells were then left untreated or treated with poly(I:C) (2 μg/ml) for 4 h ( b ) or infected with (+) IAV H1N1 at MOI of 3 for 24 h ( c ) in the presence or absence of caspase-8 inhibitor Z-IETD-FMK (IETD-8, 20 μM), caspace-9 inhibitor Z-LEHD-FMK (LEHD-9, 20 μM), or a general caspase inhibitor Z-VAD-FMK (Z-VAD, 20 μM), and cell death rate was assessed as in Figure 7 . All data are means ± S.E. of triplicates. *p

    Journal: Scientific Reports

    Article Title: Transcription Factor Runx3 Is Induced by Influenza A Virus and Double-Strand RNA and Mediates Airway Epithelial Cell Apoptosis

    doi: 10.1038/srep17916

    Figure Lengend Snippet: Both the extrinsic and intrinsic pathways are involved in Runx3-promoted cell apoptosis by dsRNA poly(I:C) and IAV infection. ( a ) Runx3 is required for the activation of caspase-8 and caspsas-9 by poly(I:C). BEAS-2B cells were transfected with 20 nM control siRNA (Con.), Runx3 siRNA-1 (Runx3-S1), or Runx3 siRNA-2 (Runx3-S2), grown for 72 h, then treated with high molecular weight poly(I:C) (2 μg/ml) for 0, 2 or 4 h. Equal amounts of cell lysates were subjected to Western blotting with specific antibodies against cleaved caspase-8, cleaved caspase-9, Runx3 or action as indicated. ( b,c ) BEAS-2B cells were infected with recombinant adenovirus containing vector alone or Runx3 and grown for 60 h. The cells were then left untreated or treated with poly(I:C) (2 μg/ml) for 4 h ( b ) or infected with (+) IAV H1N1 at MOI of 3 for 24 h ( c ) in the presence or absence of caspase-8 inhibitor Z-IETD-FMK (IETD-8, 20 μM), caspace-9 inhibitor Z-LEHD-FMK (LEHD-9, 20 μM), or a general caspase inhibitor Z-VAD-FMK (Z-VAD, 20 μM), and cell death rate was assessed as in Figure 7 . All data are means ± S.E. of triplicates. *p

    Article Snippet: TLR ligands Type B CpG oligonucleotide (ODN 2006), low and high molecular weight poly(I:C), R837, CL075, and Pam3CSK4 were from Invivo Gen. Recombinant TGFβ, TNFα, and specific peptide inhibitors for caspase-3 (Z-VAD-FMK), caspase-8 (Z-IETD-FMK) and caspase-9 (Z-LEHD-FMK) were from R & D Systems.

    Techniques: Infection, Activation Assay, Transfection, Molecular Weight, Western Blot, Recombinant, Plasmid Preparation

    Intracellular FGF1 WT protects SH-SY5Y cells from p53-dependent apoptosis. Stably transfected SH-SY5Y cells with either FGF1 WT or empty (mock) expression vectors were treated or not with etoposide for 16 h. ( a ) Polyclonal transfected SH-SY5Y apoptotic cells were characterized by flow cytometry after DiOC 6 (3) and PI staining (apoptotic cells are the DIOC−, PI− and size− cells). The graph represents the mean±S.E.M. of three independent experiments. Student’s t -tests were performed relative to the mock control, except where indicated ( n =3; n.s.: P > 0.05; ***: P ⩽0.001). ( b ) FGF1 expression and p53 activation (phosphorylated Ser15) were assessed by western blot in either polyclonal transfected cells (left panel) or isolated transfected cell lines (right panel). Actin detection was used as control. ( c ) FGF1 expression, caspase-9 and -3 cleavages were assessed in polyclonal transfected cells by western blot. Actin detection was used as control

    Journal: Cell Death & Disease

    Article Title: FGF1 protects neuroblastoma SH-SY5Y cells from p53-dependent apoptosis through an intracrine pathway regulated by FGF1 phosphorylation

    doi: 10.1038/cddis.2017.404

    Figure Lengend Snippet: Intracellular FGF1 WT protects SH-SY5Y cells from p53-dependent apoptosis. Stably transfected SH-SY5Y cells with either FGF1 WT or empty (mock) expression vectors were treated or not with etoposide for 16 h. ( a ) Polyclonal transfected SH-SY5Y apoptotic cells were characterized by flow cytometry after DiOC 6 (3) and PI staining (apoptotic cells are the DIOC−, PI− and size− cells). The graph represents the mean±S.E.M. of three independent experiments. Student’s t -tests were performed relative to the mock control, except where indicated ( n =3; n.s.: P > 0.05; ***: P ⩽0.001). ( b ) FGF1 expression and p53 activation (phosphorylated Ser15) were assessed by western blot in either polyclonal transfected cells (left panel) or isolated transfected cell lines (right panel). Actin detection was used as control. ( c ) FGF1 expression, caspase-9 and -3 cleavages were assessed in polyclonal transfected cells by western blot. Actin detection was used as control

    Article Snippet: The primary antibodies used in this study were: anti-FGF1 (AB-32-NA, R & D Systems), anti-His tag (A00186, GenScript, Piscataway, NJ, USA), anti-P-p53 (Ser-15) (9284S, Cell Signaling, Danvers, MA, USA), anti-PUMAα (N-19, Santa Cruz, Dallas, TX, USA), anti-Caspase-9 (5B4, Abcam, Cambridge, UK), anti-cleaved Caspase-3 (Asp175, Cell Signaling), anti-PARP (9542S, Cell Signaling) and anti-Actin (A2066, Sigma-Aldrich).

    Techniques: Stable Transfection, Transfection, Expressing, Flow Cytometry, Cytometry, Staining, Activation Assay, Western Blot, Isolation

    Extracellular FGF1 does not protect N2a cells from p53-dependent apoptosis. ( a ) N2a cells were pretreated or not by adding recombinant FGF1 and heparin in the culture medium (rFGF1) for 48 h, then treated or not with etoposide (Eto) for 24 h. N2a apoptotic cells were characterized by flow cytometry after DiOC 6 (3) and PI staining (apoptotic cells are the DIOC−, PI− and size− cells). The graph represents the mean±S.E.M. of three independent experiments. Student’s t -tests were performed relative to control cells, except where indicated ( n =3; n.s.: P > 0.05; ***: P ⩽0.001). ( b ) N2a cells were pretreated or not with recombinant FGF1 (rFGF1) for 48 h, and then treated or not with etoposide (Eto) for 24 h. Twenty micrograms of the corresponding cell lysate proteins were used to analyze by western blot the levels of P-p53 (Ser15) that reveals p53 activation, of pro- and cleaved caspase-9 forms and cleaved caspase-3. Actin detection was used as a control

    Journal: Cell Death & Disease

    Article Title: FGF1 protects neuroblastoma SH-SY5Y cells from p53-dependent apoptosis through an intracrine pathway regulated by FGF1 phosphorylation

    doi: 10.1038/cddis.2017.404

    Figure Lengend Snippet: Extracellular FGF1 does not protect N2a cells from p53-dependent apoptosis. ( a ) N2a cells were pretreated or not by adding recombinant FGF1 and heparin in the culture medium (rFGF1) for 48 h, then treated or not with etoposide (Eto) for 24 h. N2a apoptotic cells were characterized by flow cytometry after DiOC 6 (3) and PI staining (apoptotic cells are the DIOC−, PI− and size− cells). The graph represents the mean±S.E.M. of three independent experiments. Student’s t -tests were performed relative to control cells, except where indicated ( n =3; n.s.: P > 0.05; ***: P ⩽0.001). ( b ) N2a cells were pretreated or not with recombinant FGF1 (rFGF1) for 48 h, and then treated or not with etoposide (Eto) for 24 h. Twenty micrograms of the corresponding cell lysate proteins were used to analyze by western blot the levels of P-p53 (Ser15) that reveals p53 activation, of pro- and cleaved caspase-9 forms and cleaved caspase-3. Actin detection was used as a control

    Article Snippet: The primary antibodies used in this study were: anti-FGF1 (AB-32-NA, R & D Systems), anti-His tag (A00186, GenScript, Piscataway, NJ, USA), anti-P-p53 (Ser-15) (9284S, Cell Signaling, Danvers, MA, USA), anti-PUMAα (N-19, Santa Cruz, Dallas, TX, USA), anti-Caspase-9 (5B4, Abcam, Cambridge, UK), anti-cleaved Caspase-3 (Asp175, Cell Signaling), anti-PARP (9542S, Cell Signaling) and anti-Actin (A2066, Sigma-Aldrich).

    Techniques: Recombinant, Flow Cytometry, Cytometry, Staining, Western Blot, Activation Assay

    Role of extra- and intracellular FGF1 in neuronal SH-SY5Y, N2a and PC12 cells. ( a ) Both extra- and intra-cellular FGF1 protect human SH-SY5Y and rat PC12 cells from p53-dependent apoptosis (left). Endogenous fgf1 expression is increased by rFGF1 addition (top left). In contrast, extra- and intra-cellular FGF1 show no anti-apoptotic activity, and rFGF1 addition does not increase endogenous fgf1 expression in murine N2a cells (right). ( b ) Overexpression of intracellular FGF1 inhibits etoposide-induced apoptosis in human SH-SY5Y and rat PC12 cells by decreasing PUMA transactivation, mitochondrial membrane depolarization and permeabilization, and activation of caspase-9, caspase-3 as assessed by the cleavage of its substrate PARP (left). On the contrary, FGF1 overexpression does not protect N2a cells from etoposide-induced apoptosis, and modifies neither p53 activation, mitochondrial depolarization and permeabilization, nor cleavage of caspase-3 (right). ( c ) In human SH-SY5Y and rat PC12 cells, overexpression of wild-type FGF1 or non-phosphorylable FGF1 S130A inhibits p53-dependent apoptosis, while overexpression of the phosphomimetic FGF1 S130D or the FGF1 K132E mutant does not. Therefore, phosphorylation of FGF1 inhibits its anti-apoptotic activity in SH-SY5Y and PC12 cells. This figure compiles the results of the present study performed in neuroblastoma SH-SY5Y and N2a cells and of previous studies performed in pheochromocytoma PC12 cells 14 , 15 , 32

    Journal: Cell Death & Disease

    Article Title: FGF1 protects neuroblastoma SH-SY5Y cells from p53-dependent apoptosis through an intracrine pathway regulated by FGF1 phosphorylation

    doi: 10.1038/cddis.2017.404

    Figure Lengend Snippet: Role of extra- and intracellular FGF1 in neuronal SH-SY5Y, N2a and PC12 cells. ( a ) Both extra- and intra-cellular FGF1 protect human SH-SY5Y and rat PC12 cells from p53-dependent apoptosis (left). Endogenous fgf1 expression is increased by rFGF1 addition (top left). In contrast, extra- and intra-cellular FGF1 show no anti-apoptotic activity, and rFGF1 addition does not increase endogenous fgf1 expression in murine N2a cells (right). ( b ) Overexpression of intracellular FGF1 inhibits etoposide-induced apoptosis in human SH-SY5Y and rat PC12 cells by decreasing PUMA transactivation, mitochondrial membrane depolarization and permeabilization, and activation of caspase-9, caspase-3 as assessed by the cleavage of its substrate PARP (left). On the contrary, FGF1 overexpression does not protect N2a cells from etoposide-induced apoptosis, and modifies neither p53 activation, mitochondrial depolarization and permeabilization, nor cleavage of caspase-3 (right). ( c ) In human SH-SY5Y and rat PC12 cells, overexpression of wild-type FGF1 or non-phosphorylable FGF1 S130A inhibits p53-dependent apoptosis, while overexpression of the phosphomimetic FGF1 S130D or the FGF1 K132E mutant does not. Therefore, phosphorylation of FGF1 inhibits its anti-apoptotic activity in SH-SY5Y and PC12 cells. This figure compiles the results of the present study performed in neuroblastoma SH-SY5Y and N2a cells and of previous studies performed in pheochromocytoma PC12 cells 14 , 15 , 32

    Article Snippet: The primary antibodies used in this study were: anti-FGF1 (AB-32-NA, R & D Systems), anti-His tag (A00186, GenScript, Piscataway, NJ, USA), anti-P-p53 (Ser-15) (9284S, Cell Signaling, Danvers, MA, USA), anti-PUMAα (N-19, Santa Cruz, Dallas, TX, USA), anti-Caspase-9 (5B4, Abcam, Cambridge, UK), anti-cleaved Caspase-3 (Asp175, Cell Signaling), anti-PARP (9542S, Cell Signaling) and anti-Actin (A2066, Sigma-Aldrich).

    Techniques: Expressing, Activity Assay, Over Expression, Activation Assay, Mutagenesis

    Extracellular FGF1 protects SH-SY5Y cells from p53-dependent apoptosis. ( a ) SH-SY5Y cells were pretreated or not by adding recombinant FGF1 and heparin for 48 h (rFGF1) in the culture medium, then cells were treated or not with etoposide for 24 h (Eto). Cell survival was analyzed by crystal violet nuclei staining. ( b ) Following the same treatments, SH-SY5Y apoptotic cells were characterized by flow cytometry after DiOC 6 (3) and PI staining. Apoptotic cells correspond to the low DiOC 6 (3) (low ΔΨm, noted DIOC−) and low PI (to exclude necrotic cells, noted PI−) staining and small-sized cells (a hallmark of apoptotic cell condensation, noted size−). For ( a and b ), the graphs represent the mean±S.E.M. of three independent experiments. Student’s t -tests were performed relative to the control cells, except where indicated ( n =3; n.s.: P > 0.05; ** P ⩽0.01; *** P ⩽ 0.001). ( c ) SH-SY5Y cells were pretreated or not with recombinant FGF1 (rFGF1) for 48 h, and then treated or not with etoposide (Eto) for 6 h or 17 h. Twenty micrograms of the corresponding cell lysate proteins were used to analyze by western blot the levels of P-p53 (Ser15) that reveals p53 activation, of the p53 proapoptotic target PUMA, of pro- and cleaved caspase-9 forms and cleaved caspase-3. Actin detection was used as a control

    Journal: Cell Death & Disease

    Article Title: FGF1 protects neuroblastoma SH-SY5Y cells from p53-dependent apoptosis through an intracrine pathway regulated by FGF1 phosphorylation

    doi: 10.1038/cddis.2017.404

    Figure Lengend Snippet: Extracellular FGF1 protects SH-SY5Y cells from p53-dependent apoptosis. ( a ) SH-SY5Y cells were pretreated or not by adding recombinant FGF1 and heparin for 48 h (rFGF1) in the culture medium, then cells were treated or not with etoposide for 24 h (Eto). Cell survival was analyzed by crystal violet nuclei staining. ( b ) Following the same treatments, SH-SY5Y apoptotic cells were characterized by flow cytometry after DiOC 6 (3) and PI staining. Apoptotic cells correspond to the low DiOC 6 (3) (low ΔΨm, noted DIOC−) and low PI (to exclude necrotic cells, noted PI−) staining and small-sized cells (a hallmark of apoptotic cell condensation, noted size−). For ( a and b ), the graphs represent the mean±S.E.M. of three independent experiments. Student’s t -tests were performed relative to the control cells, except where indicated ( n =3; n.s.: P > 0.05; ** P ⩽0.01; *** P ⩽ 0.001). ( c ) SH-SY5Y cells were pretreated or not with recombinant FGF1 (rFGF1) for 48 h, and then treated or not with etoposide (Eto) for 6 h or 17 h. Twenty micrograms of the corresponding cell lysate proteins were used to analyze by western blot the levels of P-p53 (Ser15) that reveals p53 activation, of the p53 proapoptotic target PUMA, of pro- and cleaved caspase-9 forms and cleaved caspase-3. Actin detection was used as a control

    Article Snippet: The primary antibodies used in this study were: anti-FGF1 (AB-32-NA, R & D Systems), anti-His tag (A00186, GenScript, Piscataway, NJ, USA), anti-P-p53 (Ser-15) (9284S, Cell Signaling, Danvers, MA, USA), anti-PUMAα (N-19, Santa Cruz, Dallas, TX, USA), anti-Caspase-9 (5B4, Abcam, Cambridge, UK), anti-cleaved Caspase-3 (Asp175, Cell Signaling), anti-PARP (9542S, Cell Signaling) and anti-Actin (A2066, Sigma-Aldrich).

    Techniques: Recombinant, Staining, Flow Cytometry, Cytometry, Western Blot, Activation Assay

    Translocation of caspase-9 from mitochondria to nuclei in PC12 cells during apoptosis. PC12-Puro ( a – h ) or PC12-Bcl-2 ( i – l ) cells were cultured with 25 nM Mitotracker without ( a – d ) or with ( e – l ) addition of 150 μM tamoxifen for 12 ( e – h ) or 24 hr ( i – l ). Cells were fixed, permeabilized, and incubated with anti-caspase-9 antiserum followed by FITC-conjugated secondary antibody. In some cases, the anti-caspase-9 antiserum was preadsorbed with recombinant caspase-9 protein d and l ) by using filters to selectively reveal FITC-labeled caspase-9 ( a , e , and i ), Mitotracker ( b , f , and j ), or both colors ( c , d , g , h , k , and l ). In m , PC12-Puro cells were cultured with or without 3 μM staurosporine or 150 μM tamoxifen for 12 hr. Cell lysates were prepared, were normalized for total protein content, and were analyzed by SDS/PAGE/immunoblotting by using anti-caspase-9 antiserum Bur49. Note that this antiserum preferentially reacts with the small subunit of the processed protease.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Release of caspase-9 from mitochondria during neuronal apoptosis and cerebral ischemia

    doi:

    Figure Lengend Snippet: Translocation of caspase-9 from mitochondria to nuclei in PC12 cells during apoptosis. PC12-Puro ( a – h ) or PC12-Bcl-2 ( i – l ) cells were cultured with 25 nM Mitotracker without ( a – d ) or with ( e – l ) addition of 150 μM tamoxifen for 12 ( e – h ) or 24 hr ( i – l ). Cells were fixed, permeabilized, and incubated with anti-caspase-9 antiserum followed by FITC-conjugated secondary antibody. In some cases, the anti-caspase-9 antiserum was preadsorbed with recombinant caspase-9 protein d and l ) by using filters to selectively reveal FITC-labeled caspase-9 ( a , e , and i ), Mitotracker ( b , f , and j ), or both colors ( c , d , g , h , k , and l ). In m , PC12-Puro cells were cultured with or without 3 μM staurosporine or 150 μM tamoxifen for 12 hr. Cell lysates were prepared, were normalized for total protein content, and were analyzed by SDS/PAGE/immunoblotting by using anti-caspase-9 antiserum Bur49. Note that this antiserum preferentially reacts with the small subunit of the processed protease.

    Article Snippet: PC12 cells were fixed in 4% PFA in PBS (pH 7.4), were permeabilized with 0.2% Triton X-100, and were subjected to immunostaining using anti-caspase-9 antiserum with FITC-conjugated goat anti-rabbit IgG (Sigma) essentially as described ( ).

    Techniques: Translocation Assay, Cell Culture, Incubation, Recombinant, Labeling, SDS Page

    Immunohistochemical analysis of Caspase-9 in tissues. Selected examples of cells that exhibited an organellar pattern of caspase-9 immunostaining are presented. Results were confirmed by immunostaining with at least two independent anti-caspase-9 antisera [Bur73 ( a – c ); Bur49 ( d – k )]. Controls for nonspecific immunostaining also were performed for all tissues, including use of preimmune serum and antigen-preadsorbed serum, though only two examples are presented here ( f and i ). a – c demonstrate caspase-9 immunoreactivity in the brain, including large and small striatal neurons ( a – b , ×400) and brain stem motorneurons ( c , ×1,000). ( d and e ) caspase-9 immunopositive presynaptic termini synapsing on large cortical neurons that are caspase-9 immunonegative (×1,000). [Immuno-EM analysis indicated that presynaptic mitochondria account for circumferential immunostaining surrounding such cells (data not shown).] ( f ) Antigen preadsorbed antiserum yields essentially no immunostaining in brain stem (×400). ( g ) Caspase-9 immunostaining in rat skeletal muscle (×400). Rat heart stained with antibody Bur-49 ( h , ×400) or with antibody that had been preadsorbed with caspase 9 recombinant protein ( i , ×400). ( j ) Skeletal muscle (×200) derived from a caspase-9 knock-out mouse, revealing essentially no caspase-9 immunostaining. Age-matched tissue from normal littermates was caspase-9 immunopositive (data not shown). ( k ) Distal convoluted tubules of rat kidney, revealing a combination of cytosolic (dark arrows) and organellar caspase-9 immunostaining (×1,000).

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Release of caspase-9 from mitochondria during neuronal apoptosis and cerebral ischemia

    doi:

    Figure Lengend Snippet: Immunohistochemical analysis of Caspase-9 in tissues. Selected examples of cells that exhibited an organellar pattern of caspase-9 immunostaining are presented. Results were confirmed by immunostaining with at least two independent anti-caspase-9 antisera [Bur73 ( a – c ); Bur49 ( d – k )]. Controls for nonspecific immunostaining also were performed for all tissues, including use of preimmune serum and antigen-preadsorbed serum, though only two examples are presented here ( f and i ). a – c demonstrate caspase-9 immunoreactivity in the brain, including large and small striatal neurons ( a – b , ×400) and brain stem motorneurons ( c , ×1,000). ( d and e ) caspase-9 immunopositive presynaptic termini synapsing on large cortical neurons that are caspase-9 immunonegative (×1,000). [Immuno-EM analysis indicated that presynaptic mitochondria account for circumferential immunostaining surrounding such cells (data not shown).] ( f ) Antigen preadsorbed antiserum yields essentially no immunostaining in brain stem (×400). ( g ) Caspase-9 immunostaining in rat skeletal muscle (×400). Rat heart stained with antibody Bur-49 ( h , ×400) or with antibody that had been preadsorbed with caspase 9 recombinant protein ( i , ×400). ( j ) Skeletal muscle (×200) derived from a caspase-9 knock-out mouse, revealing essentially no caspase-9 immunostaining. Age-matched tissue from normal littermates was caspase-9 immunopositive (data not shown). ( k ) Distal convoluted tubules of rat kidney, revealing a combination of cytosolic (dark arrows) and organellar caspase-9 immunostaining (×1,000).

    Article Snippet: PC12 cells were fixed in 4% PFA in PBS (pH 7.4), were permeabilized with 0.2% Triton X-100, and were subjected to immunostaining using anti-caspase-9 antiserum with FITC-conjugated goat anti-rabbit IgG (Sigma) essentially as described ( ).

    Techniques: Immunohistochemistry, Immunostaining, Staining, Recombinant, Derivative Assay, Knock-Out

    Immuno-EM analysis of Caspase-9 in rat heart, brain, and isolated mitochondria. Ultrathin sections of heart muscle ( a and b ) or cerebellum ( c and d ) were analyzed by using postembedding immunogold methods and anti-caspase-9 antiserum Bur49. Images in c and d represent presynaptic mitochondria. Use of anti-caspase 9 antiserum preadsorbed with recombinant caspase-9 protein resulted in essentially no immunogold staining (data not shown). In e – h , mitochondria were isolated from rat heart and were fixed, embedded, and sectioned for immunogold EM analysis using anti-caspase-9 antisera ( e and f ) or anti-caspase-9 antiserum preadsorbed with recombinant caspase-9 protein ( g ) or stained with monoclonal antibody to Hsp60 ( h ). Identical staining procedures performed by using an irrelevant mouse IgG as a control for Hsp60 produced negligible gold deposits.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Release of caspase-9 from mitochondria during neuronal apoptosis and cerebral ischemia

    doi:

    Figure Lengend Snippet: Immuno-EM analysis of Caspase-9 in rat heart, brain, and isolated mitochondria. Ultrathin sections of heart muscle ( a and b ) or cerebellum ( c and d ) were analyzed by using postembedding immunogold methods and anti-caspase-9 antiserum Bur49. Images in c and d represent presynaptic mitochondria. Use of anti-caspase 9 antiserum preadsorbed with recombinant caspase-9 protein resulted in essentially no immunogold staining (data not shown). In e – h , mitochondria were isolated from rat heart and were fixed, embedded, and sectioned for immunogold EM analysis using anti-caspase-9 antisera ( e and f ) or anti-caspase-9 antiserum preadsorbed with recombinant caspase-9 protein ( g ) or stained with monoclonal antibody to Hsp60 ( h ). Identical staining procedures performed by using an irrelevant mouse IgG as a control for Hsp60 produced negligible gold deposits.

    Article Snippet: PC12 cells were fixed in 4% PFA in PBS (pH 7.4), were permeabilized with 0.2% Triton X-100, and were subjected to immunostaining using anti-caspase-9 antiserum with FITC-conjugated goat anti-rabbit IgG (Sigma) essentially as described ( ).

    Techniques: Isolation, Recombinant, Staining, Produced

    Pro-caspase-9 release from isolated mitochondria is induced by digitonin, Ca 2+ , or Bax. ( A ) Isotonic, detergent-free rat brain homogenates were used to prepare an organelle-containing (P) fraction and a cytosolic supernatant (S) fraction. The P and S fractions (50 μg) were subjected to immunoblot analysis by using anti-caspase-9 antiserum Bur49. Recombinant caspase-9 (15 ng) was included as a control. Reprobing the same blot with antibodies specific for mitochondrial and cytosolic proteins verified appropriate fractionation (data not shown). ( B – F ), were normalized for total protein content and were subjected to various treatments as indicated, then were sedimented by centrifugation at 10,000 × g for 4 min to collect the post-mitochondria supernatant (S) or pellet (P) fractions. Samples were analyzed by SDS/PAGE/immunoblotting by using various antibodies with enhanced chemiluminescence-based detection. ( B ) Mitochondria were incubated in either isotonic (ISO) or hypotonic (HYPO) solution for 20 min at 4°C before preparing S and P fractions for immunoblot analysis with antibodies to caspase-9 and Hsp60. Recombinant caspase-9 was included as a control. ( C ) Mitochondria were incubated in isotonic solution containing 1% digitonin at 4°C for 20 min and then were sedimented. S and P fractions were analyzed by using antibodies to caspase-9, cyto-c and COX-IV. ( D ) Mitochondria were incubated at 4°C for 20 min with (+) or without (−) 125 ng/ml trypsin; then, the entire contents were mixed with an equal volume of Laemmli loading solution and were subjected to SDS/PAGE using antibodies specific for caspase-9, cyto-c, or Bcl-X L . ( E and F ) Mitochondria were treated in the absence of EGTA with either 25 μM CaCl 2 or 1.2 μM Bax at 30°C for 45 min and then were sedimented to produce S and P fractions. S fractions were analyzed by immunoblotting for caspase-9 ( Top ), cyto-c ( Middle ), or Hsp60, which was negative (data not shown). Immunoblot analysis of pellet fractions using anti-Hsp60 antibody confirmed input of equivalent amounts of mitochondria for all samples ( Bottom ).

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Release of caspase-9 from mitochondria during neuronal apoptosis and cerebral ischemia

    doi:

    Figure Lengend Snippet: Pro-caspase-9 release from isolated mitochondria is induced by digitonin, Ca 2+ , or Bax. ( A ) Isotonic, detergent-free rat brain homogenates were used to prepare an organelle-containing (P) fraction and a cytosolic supernatant (S) fraction. The P and S fractions (50 μg) were subjected to immunoblot analysis by using anti-caspase-9 antiserum Bur49. Recombinant caspase-9 (15 ng) was included as a control. Reprobing the same blot with antibodies specific for mitochondrial and cytosolic proteins verified appropriate fractionation (data not shown). ( B – F ), were normalized for total protein content and were subjected to various treatments as indicated, then were sedimented by centrifugation at 10,000 × g for 4 min to collect the post-mitochondria supernatant (S) or pellet (P) fractions. Samples were analyzed by SDS/PAGE/immunoblotting by using various antibodies with enhanced chemiluminescence-based detection. ( B ) Mitochondria were incubated in either isotonic (ISO) or hypotonic (HYPO) solution for 20 min at 4°C before preparing S and P fractions for immunoblot analysis with antibodies to caspase-9 and Hsp60. Recombinant caspase-9 was included as a control. ( C ) Mitochondria were incubated in isotonic solution containing 1% digitonin at 4°C for 20 min and then were sedimented. S and P fractions were analyzed by using antibodies to caspase-9, cyto-c and COX-IV. ( D ) Mitochondria were incubated at 4°C for 20 min with (+) or without (−) 125 ng/ml trypsin; then, the entire contents were mixed with an equal volume of Laemmli loading solution and were subjected to SDS/PAGE using antibodies specific for caspase-9, cyto-c, or Bcl-X L . ( E and F ) Mitochondria were treated in the absence of EGTA with either 25 μM CaCl 2 or 1.2 μM Bax at 30°C for 45 min and then were sedimented to produce S and P fractions. S fractions were analyzed by immunoblotting for caspase-9 ( Top ), cyto-c ( Middle ), or Hsp60, which was negative (data not shown). Immunoblot analysis of pellet fractions using anti-Hsp60 antibody confirmed input of equivalent amounts of mitochondria for all samples ( Bottom ).

    Article Snippet: PC12 cells were fixed in 4% PFA in PBS (pH 7.4), were permeabilized with 0.2% Triton X-100, and were subjected to immunostaining using anti-caspase-9 antiserum with FITC-conjugated goat anti-rabbit IgG (Sigma) essentially as described ( ).

    Techniques: Isolation, Recombinant, Fractionation, Centrifugation, SDS Page, Incubation

    ). ( a and b ) Typical organellar caspase-9 immunostaining is shown in CA1-CA2 sector hippocampal neurons of a sham-operated animal ( a , ×150; b , ×1,000); ( c ) At 2 hr after ischemia/reperfusion in the CA1–2 region of hippocampus, combinations of normal-appearing neurons (open arrows) that retain mitochondrial pattern of caspase-9 immunostaining are seen adjacent to injured neurons (dark arrows) in which caspase-9 immunostaining is diffuse or accumulated around nuclei (×200). ( d ) At 24 hr, many degenerating neurons contain nuclear caspase-9 (×1,000). ( e ) These degenerating neurons at 24 hr are mostly TUNEL-positive (black arrows) (×1,000). ( f and g ) Large cortical neurons of a sham-operated animal immunostained with anti-caspase-9 antiserum without ( f ) or with ( g ) prior preabsorption with competing caspase-9 protein (×200). ( h ) At 2 hr after ischemia/reperfusion, combinations of normal (open arrows) and degenerating (dark arrows) cortical neurons are observed, with caspase 9 immunoreactivity localized diffusely over the cytosol and nuclei of degenerating neurons (×400). ( i ) At 2 hr after ischemia, most degenerating neurons contain nuclear caspase-9 immunostaining (brown) (open arrows) but are TUNEL-negative. Occasional cells (black arrow) exhibit double labeling with caspase 9 (brown) and TUNEL (purple), with TUNEL-labeling dominating over caspase 9 brown staining (×1,000).

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Release of caspase-9 from mitochondria during neuronal apoptosis and cerebral ischemia

    doi:

    Figure Lengend Snippet: ). ( a and b ) Typical organellar caspase-9 immunostaining is shown in CA1-CA2 sector hippocampal neurons of a sham-operated animal ( a , ×150; b , ×1,000); ( c ) At 2 hr after ischemia/reperfusion in the CA1–2 region of hippocampus, combinations of normal-appearing neurons (open arrows) that retain mitochondrial pattern of caspase-9 immunostaining are seen adjacent to injured neurons (dark arrows) in which caspase-9 immunostaining is diffuse or accumulated around nuclei (×200). ( d ) At 24 hr, many degenerating neurons contain nuclear caspase-9 (×1,000). ( e ) These degenerating neurons at 24 hr are mostly TUNEL-positive (black arrows) (×1,000). ( f and g ) Large cortical neurons of a sham-operated animal immunostained with anti-caspase-9 antiserum without ( f ) or with ( g ) prior preabsorption with competing caspase-9 protein (×200). ( h ) At 2 hr after ischemia/reperfusion, combinations of normal (open arrows) and degenerating (dark arrows) cortical neurons are observed, with caspase 9 immunoreactivity localized diffusely over the cytosol and nuclei of degenerating neurons (×400). ( i ) At 2 hr after ischemia, most degenerating neurons contain nuclear caspase-9 immunostaining (brown) (open arrows) but are TUNEL-negative. Occasional cells (black arrow) exhibit double labeling with caspase 9 (brown) and TUNEL (purple), with TUNEL-labeling dominating over caspase 9 brown staining (×1,000).

    Article Snippet: PC12 cells were fixed in 4% PFA in PBS (pH 7.4), were permeabilized with 0.2% Triton X-100, and were subjected to immunostaining using anti-caspase-9 antiserum with FITC-conjugated goat anti-rabbit IgG (Sigma) essentially as described ( ).

    Techniques: Immunostaining, TUNEL Assay, Labeling, Staining

    Characterization of anti-caspase-9 antisera. Antisera were generated in rabbits by using either purified recombinant processed caspase-9 (Bur49) or a synthetic peptide corresponding to amino acids 112–130 of human caspase-9 (Bur 73). ( A and B ). Note that antiserum Bur49 reacts primarily with the ≈15-kDa small subunit whereas antiserum Bur73 reacts with the ≈35-kDa large subunit of caspase-9. Both antibodies equally recognize the proform of caspase-9 (data not shown). ( C ) Tissue lysates were prepared from human autopsy material, were normalized for total protein (100 μg), and were subjected to SDS/PAGE/immunoblot assay by using anti-caspase-9 antiserum Bur73. Most tissues contain the unprocessed ≈50-kDa pro-caspase-9 protein whereas processed caspase-9 was identified in brain and peripheral nerve tissue samples.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Release of caspase-9 from mitochondria during neuronal apoptosis and cerebral ischemia

    doi:

    Figure Lengend Snippet: Characterization of anti-caspase-9 antisera. Antisera were generated in rabbits by using either purified recombinant processed caspase-9 (Bur49) or a synthetic peptide corresponding to amino acids 112–130 of human caspase-9 (Bur 73). ( A and B ). Note that antiserum Bur49 reacts primarily with the ≈15-kDa small subunit whereas antiserum Bur73 reacts with the ≈35-kDa large subunit of caspase-9. Both antibodies equally recognize the proform of caspase-9 (data not shown). ( C ) Tissue lysates were prepared from human autopsy material, were normalized for total protein (100 μg), and were subjected to SDS/PAGE/immunoblot assay by using anti-caspase-9 antiserum Bur73. Most tissues contain the unprocessed ≈50-kDa pro-caspase-9 protein whereas processed caspase-9 was identified in brain and peripheral nerve tissue samples.

    Article Snippet: PC12 cells were fixed in 4% PFA in PBS (pH 7.4), were permeabilized with 0.2% Triton X-100, and were subjected to immunostaining using anti-caspase-9 antiserum with FITC-conjugated goat anti-rabbit IgG (Sigma) essentially as described ( ).

    Techniques: Generated, Purification, Recombinant, SDS Page

    Effect of psoralidin on caspase signaling and viability of AIPC cells (A). PC-3 and DU-145 cells (70–80% confluency) were treated with 60 and 45 μM psoralidin, respectively, for 12 and 24 h, and Western blot analysis was performed using caspase-9, caspase-8, caspase -3 and PARP antibodies. Actin was used as the internal loading control. (B). PC-3 and DU-145 cells (70–80% confluency) were treated with psoralidin alone, caspase inhibitors alone (3 or 9) and a combination of caspase inhibitors and psoralidin, and a fluorometric assay was performed to determine caspase-3 activation. Bars represent fold increase in caspase activity in each treatment group. (C). PC-3, DU-145 and PzHPv-7 (70–80% confluency) cells were treated with varying concentrations of psoralidin, and cell viability was determined using Trypan blue assay. A dose-response curve was plotted, and each data point represents mean percentage of cell viability±SD.

    Journal: Apoptosis : an international journal on programmed cell death

    Article Title: Inhibiting TNF-mediated signaling: a novel therapeutic paradigm for androgen independent prostate cancer

    doi: 10.1007/s10495-009-0416-9

    Figure Lengend Snippet: Effect of psoralidin on caspase signaling and viability of AIPC cells (A). PC-3 and DU-145 cells (70–80% confluency) were treated with 60 and 45 μM psoralidin, respectively, for 12 and 24 h, and Western blot analysis was performed using caspase-9, caspase-8, caspase -3 and PARP antibodies. Actin was used as the internal loading control. (B). PC-3 and DU-145 cells (70–80% confluency) were treated with psoralidin alone, caspase inhibitors alone (3 or 9) and a combination of caspase inhibitors and psoralidin, and a fluorometric assay was performed to determine caspase-3 activation. Bars represent fold increase in caspase activity in each treatment group. (C). PC-3, DU-145 and PzHPv-7 (70–80% confluency) cells were treated with varying concentrations of psoralidin, and cell viability was determined using Trypan blue assay. A dose-response curve was plotted, and each data point represents mean percentage of cell viability±SD.

    Article Snippet: Human recombinant TNF-α (hr TNF-α) and Caspase-9 inhibitor were purchased from Calbiochem (Gibbstown, NJ) and used at a concentration of 10 ng/ml and 10 μM, respectively.

    Techniques: Western Blot, Activation Assay, Activity Assay

    Role of caspases in LtxA-mediated cell death. CEM (A), Jurkat (B) and RL (C) cells were pretreated with the pancaspase inhibitor z-VAD-FMK (50 μM) for 1 hour and subsequently treated with LtxA for 24 hours. Cytotoxity was determined by flow cytometry, and the percent cell death is expressed as the sum of annexin V + /7-AAD − and annexin V + /7-AAD + cells. Data represent the average of three independent experiments. Error bars show SEM. The significance of the differences was determined using a Student’s t -test. *p≤0.05; ** p≤0.01. (D–G) Caspase-8 and -9 activation in Jurkat cells. Jurkat cells were treated with 0.2 μg/ml LtxA for various times and cleavage of caspase-8 (D, E) caspase-9 (F) or total GAPDH levels (G) was evaluated. Solid arrows represent the procaspase form and open arrows indicate the cleavage product. D) Western blot analysis of caspase-8. Lane 1, Untreated Jurkat cells; lane 2, 2 hours LtxA; lane 3, 6 hours LtxA. E) Flow cytometric analaysis of Jurkat cells treated with 0.2 μg/ml LtxA, permeabilized, and stained for caspase-8 activation. The gray peak represents unstained cells; black, untreated cells; red, 2 hours LtxA; blue, 6 hours LtxA. F) Western blot analysis of caspase-9. First lane, untreated cells; second lane, 2 hours LtxA; third lane, 6 hours LtxA; fourth lane, 4 hours staurosporine treated cells (1 μg/ml). G) Western blot analysis of GAPDH. First lane, untreated cells; second lane, 2 hours LtxA; third lane, 6 hours LtxA. (H–J) Caspase-8 and -9 activation in RL cells. RL cells were treated with 0.2 μg/ml LtxA for various times and cleavage of caspase-8 (H) or caspase-9 (I) was evaluated by western blot analysis. Solid arrows represent the procaspase form and open arrows indicate the cleavage product. H) Western blot analysis of caspase-8. First lane, untreated cells; second lane, 3 hours LtxA; third lane, 6 hours LtxA. I) Western blot analysis of caspase-9. First lane, untreated cells; second lane, 3 hours LtxA; third lane 6 hours LtxA; fourth lane, 4 hours staurosporine treated cells (1 μg/ml). J) Western blot analysis of GAPDH. First lane, untreated cells; second lane, 3 hours LtxA; third lane, 6 hours LtxA.

    Journal: Leukemia research

    Article Title: LFA-1-targeting Leukotoxin (LtxA; Leukothera®) causes lymphoma tumor regression in a humanized mouse model and requires caspase-8 and Fas to kill malignant lymphocytes*

    doi: 10.1016/j.leukres.2015.03.010

    Figure Lengend Snippet: Role of caspases in LtxA-mediated cell death. CEM (A), Jurkat (B) and RL (C) cells were pretreated with the pancaspase inhibitor z-VAD-FMK (50 μM) for 1 hour and subsequently treated with LtxA for 24 hours. Cytotoxity was determined by flow cytometry, and the percent cell death is expressed as the sum of annexin V + /7-AAD − and annexin V + /7-AAD + cells. Data represent the average of three independent experiments. Error bars show SEM. The significance of the differences was determined using a Student’s t -test. *p≤0.05; ** p≤0.01. (D–G) Caspase-8 and -9 activation in Jurkat cells. Jurkat cells were treated with 0.2 μg/ml LtxA for various times and cleavage of caspase-8 (D, E) caspase-9 (F) or total GAPDH levels (G) was evaluated. Solid arrows represent the procaspase form and open arrows indicate the cleavage product. D) Western blot analysis of caspase-8. Lane 1, Untreated Jurkat cells; lane 2, 2 hours LtxA; lane 3, 6 hours LtxA. E) Flow cytometric analaysis of Jurkat cells treated with 0.2 μg/ml LtxA, permeabilized, and stained for caspase-8 activation. The gray peak represents unstained cells; black, untreated cells; red, 2 hours LtxA; blue, 6 hours LtxA. F) Western blot analysis of caspase-9. First lane, untreated cells; second lane, 2 hours LtxA; third lane, 6 hours LtxA; fourth lane, 4 hours staurosporine treated cells (1 μg/ml). G) Western blot analysis of GAPDH. First lane, untreated cells; second lane, 2 hours LtxA; third lane, 6 hours LtxA. (H–J) Caspase-8 and -9 activation in RL cells. RL cells were treated with 0.2 μg/ml LtxA for various times and cleavage of caspase-8 (H) or caspase-9 (I) was evaluated by western blot analysis. Solid arrows represent the procaspase form and open arrows indicate the cleavage product. H) Western blot analysis of caspase-8. First lane, untreated cells; second lane, 3 hours LtxA; third lane, 6 hours LtxA. I) Western blot analysis of caspase-9. First lane, untreated cells; second lane, 3 hours LtxA; third lane 6 hours LtxA; fourth lane, 4 hours staurosporine treated cells (1 μg/ml). J) Western blot analysis of GAPDH. First lane, untreated cells; second lane, 3 hours LtxA; third lane, 6 hours LtxA.

    Article Snippet: Primary antibodies (1:1000) used were anti-human caspase-8 (clone IC12 from Cell Signaling), anti-human caspase-9 (clone LAP6 from R & D Systems), and anti-human GAPDH.

    Techniques: Flow Cytometry, Cytometry, Activation Assay, Western Blot, Staining

    The role of caspase-8 and caspase-9. RL (A) and Jurkat (B) cells were pretreated with either caspase-8 inhibitor z-IETD-FMK or caspase-9 inhibitor z-LEHD-FMK (50 μM) for 1 hour and subsequently treated with LtxA for 24 hours. Cytotoxity was determined by annexin V/7-AAD staining and flow cytometry. C) A caspase-8 mutant and wild type control A3 cells were treated with various concentrations of LtxA for 24 hours. Cytotoxity was determined by annexin V/7-AAD staining and flow cytometry. Data represent the average of three independent experiments. Error bars show SEM. The significance of the differences was determined using a Student’s t -test. ** p≤0.01; ***p≤0.001.

    Journal: Leukemia research

    Article Title: LFA-1-targeting Leukotoxin (LtxA; Leukothera®) causes lymphoma tumor regression in a humanized mouse model and requires caspase-8 and Fas to kill malignant lymphocytes*

    doi: 10.1016/j.leukres.2015.03.010

    Figure Lengend Snippet: The role of caspase-8 and caspase-9. RL (A) and Jurkat (B) cells were pretreated with either caspase-8 inhibitor z-IETD-FMK or caspase-9 inhibitor z-LEHD-FMK (50 μM) for 1 hour and subsequently treated with LtxA for 24 hours. Cytotoxity was determined by annexin V/7-AAD staining and flow cytometry. C) A caspase-8 mutant and wild type control A3 cells were treated with various concentrations of LtxA for 24 hours. Cytotoxity was determined by annexin V/7-AAD staining and flow cytometry. Data represent the average of three independent experiments. Error bars show SEM. The significance of the differences was determined using a Student’s t -test. ** p≤0.01; ***p≤0.001.

    Article Snippet: Primary antibodies (1:1000) used were anti-human caspase-8 (clone IC12 from Cell Signaling), anti-human caspase-9 (clone LAP6 from R & D Systems), and anti-human GAPDH.

    Techniques: Staining, Flow Cytometry, Cytometry, Mutagenesis

    cIAP1 Prevents Procaspase-3 Activation by the Cytochrome c-dependent Caspase-9 Apoptosome

    Journal: The Journal of Biological Chemistry

    Article Title: cIAP1 Cooperatively Inhibits Procaspase-3 Activation by the Caspase-9 Apoptosome *

    doi: 10.1074/jbc.M110.125955

    Figure Lengend Snippet: cIAP1 Prevents Procaspase-3 Activation by the Cytochrome c-dependent Caspase-9 Apoptosome

    Article Snippet: S100 or S64 proteins were size-fractionated by SDS-PAGE, transferred to polyvinylidene difluoride membrane, and immunostained with antibody to caspase-9 (mouse monoclonal MAB8301, R & D Systems) or caspase-3 (rabbit monoclonal anti-cleaved caspase-3, 8G10, Cell Signaling Technology).

    Techniques: Activation Assay

    Gadd45 α triggers Shigella -mediated apoptotic cell death in infected HeLa cells. Activity of caspase-8 ( A ), caspase-9 ( B ) and caspase-3 ( C ), and TUNEL assay ( E and F ) on HeLa cells transiently transfected with a Gadd45 α or a scramble si RNA. Cells were infected with M90T at MOI of 100 for the reported time points. HeLa cells treated with STP or with CHX plus TNF- α , as detailed in Figure 2 , were used as a control ( D ). HeLa NI, non-infected HeLa cells. Report assay data correspond to the mean±S.D. (triplicate determinations) and are representative of three independent luminometric assays. * P

    Journal: Cell Death & Disease

    Article Title: Gadd45α activity is the principal effector of Shigella mitochondria-dependent epithelial cell death in vitro and ex vivo

    doi: 10.1038/cddis.2011.4

    Figure Lengend Snippet: Gadd45 α triggers Shigella -mediated apoptotic cell death in infected HeLa cells. Activity of caspase-8 ( A ), caspase-9 ( B ) and caspase-3 ( C ), and TUNEL assay ( E and F ) on HeLa cells transiently transfected with a Gadd45 α or a scramble si RNA. Cells were infected with M90T at MOI of 100 for the reported time points. HeLa cells treated with STP or with CHX plus TNF- α , as detailed in Figure 2 , were used as a control ( D ). HeLa NI, non-infected HeLa cells. Report assay data correspond to the mean±S.D. (triplicate determinations) and are representative of three independent luminometric assays. * P

    Article Snippet: Caspase-8 and caspase-9 depletion was verified by western blot analysis with purified mouse anti-human caspase-8 monoclonal antibody (clone 3-1-9, BD Pharmingen, San José, CA, USA) and monoclonal anti-human caspase-9 (clone LAP6, R & D System), respectively.

    Techniques: Infection, Activity Assay, TUNEL Assay, Transfection

    Caspase activation in HeLa cells infected with Shigella . ( A ) Kinetics of caspase-3 activation following infection of HeLa cells with M90T at different MOIs as above. ( B ) Caspase-9 and ( C ) caspase-8 activity in HeLa cells infected with M90T and its noninvasive variant BS176 (MOI of 100) at the reported time points. HeLa cells treated for 4 h with STP (2 μ M) or with CHX (10 μ g/ml) plus TNF- α (100 ng/ml) for 12 h were used as a control of caspase-9/3 and caspase-8 activation, respectively. HeLa NI, non-infected HeLa cells. Report assay data correspond to the mean±S.D. (triplicate determinations) and are representative of three independent luminometric assays. * P

    Journal: Cell Death & Disease

    Article Title: Gadd45α activity is the principal effector of Shigella mitochondria-dependent epithelial cell death in vitro and ex vivo

    doi: 10.1038/cddis.2011.4

    Figure Lengend Snippet: Caspase activation in HeLa cells infected with Shigella . ( A ) Kinetics of caspase-3 activation following infection of HeLa cells with M90T at different MOIs as above. ( B ) Caspase-9 and ( C ) caspase-8 activity in HeLa cells infected with M90T and its noninvasive variant BS176 (MOI of 100) at the reported time points. HeLa cells treated for 4 h with STP (2 μ M) or with CHX (10 μ g/ml) plus TNF- α (100 ng/ml) for 12 h were used as a control of caspase-9/3 and caspase-8 activation, respectively. HeLa NI, non-infected HeLa cells. Report assay data correspond to the mean±S.D. (triplicate determinations) and are representative of three independent luminometric assays. * P

    Article Snippet: Caspase-8 and caspase-9 depletion was verified by western blot analysis with purified mouse anti-human caspase-8 monoclonal antibody (clone 3-1-9, BD Pharmingen, San José, CA, USA) and monoclonal anti-human caspase-9 (clone LAP6, R & D System), respectively.

    Techniques: Activation Assay, Infection, Activity Assay, Variant Assay

    Shigella -infected HeLa cells undergo intrinsic apoptosis. ( a ) FACS analysis (forward scatter, FSC, versus TMRM) showing mitochondrial depolarization. The percentages of viable cells (V, TMRM positive, in the R1 quadrant) and of depolarized cells (D, TMRM negative, in the R2 quadrant) are reported. ( b ) FACS analysis of mitochondrial potential (TMRM staining; blue population) and of caspase-3 activation (FLICA caspase-3 labeling; red population). Both populations were determined on diagrams FSC versus fluorophore, and are shown together on a FSC versus SSC plot. In ( a and b ), HeLa cells were infected with M90T (MOI 100) for the reported time points; treatment with H 2 O 2 (5 mM for 1 h) and with STP as in Figure 2 were used as positive controls of mitochondrial depolarization and of caspase activation. Cells transiently transfected with si RNA for caspase-9 ( c ) or for caspase-8 ( d ) were infected with M90T at MOI 100 and caspase-3 activity was measured at the reported time points. HeLa cells treated for 4 h with STP or with CHX plus TNF- α as specified in Figure 2 were used as a control. HeLa NI, non-infected HeLa cells. Report assay data correspond to the mean±S.D. (triplicate determinations) and are representative of five independent luminometric assays. * P

    Journal: Cell Death & Disease

    Article Title: Gadd45α activity is the principal effector of Shigella mitochondria-dependent epithelial cell death in vitro and ex vivo

    doi: 10.1038/cddis.2011.4

    Figure Lengend Snippet: Shigella -infected HeLa cells undergo intrinsic apoptosis. ( a ) FACS analysis (forward scatter, FSC, versus TMRM) showing mitochondrial depolarization. The percentages of viable cells (V, TMRM positive, in the R1 quadrant) and of depolarized cells (D, TMRM negative, in the R2 quadrant) are reported. ( b ) FACS analysis of mitochondrial potential (TMRM staining; blue population) and of caspase-3 activation (FLICA caspase-3 labeling; red population). Both populations were determined on diagrams FSC versus fluorophore, and are shown together on a FSC versus SSC plot. In ( a and b ), HeLa cells were infected with M90T (MOI 100) for the reported time points; treatment with H 2 O 2 (5 mM for 1 h) and with STP as in Figure 2 were used as positive controls of mitochondrial depolarization and of caspase activation. Cells transiently transfected with si RNA for caspase-9 ( c ) or for caspase-8 ( d ) were infected with M90T at MOI 100 and caspase-3 activity was measured at the reported time points. HeLa cells treated for 4 h with STP or with CHX plus TNF- α as specified in Figure 2 were used as a control. HeLa NI, non-infected HeLa cells. Report assay data correspond to the mean±S.D. (triplicate determinations) and are representative of five independent luminometric assays. * P

    Article Snippet: Caspase-8 and caspase-9 depletion was verified by western blot analysis with purified mouse anti-human caspase-8 monoclonal antibody (clone 3-1-9, BD Pharmingen, San José, CA, USA) and monoclonal anti-human caspase-9 (clone LAP6, R & D System), respectively.

    Techniques: Infection, FACS, Staining, Activation Assay, Labeling, Transfection, Activity Assay

    Induction of caspase-dependent apoptosis by hydralazine in leukemic T cells A. Jurkat, CEM-6, MOLT-4 and peripheral blood lymphocytes (PBL) were treated with different doses of hydralazine (40, 80, 200, 400 and 600 μM; upper panel) or procainamide (100, 250 and 500 μM; lower panel) for either 48 hr (T cell lines) or 72 hr (PBL in upper panel). B. Expression of DNTM1 was determined by Western blotting in Jurkat cells after treatment for 24 and 48 hr without (C) or with the indicated concentrations of hydralazine and procainamide. C. Jurkat cells were preincubated for 1 hr in the absence or in the presence of the caspase inhibitor Q-VD-OPh (20 μM) before treatment without (control) or with 200 and 600 μM hydralazine (H-200 and H-600, respectively) for 24 and 48 hr. D. Activation of caspases-8, -3 and -9 was determined by Western blot in Jurkat cells treated without (C) or with 80, 200 and 600 μM hydralazine (H-80, H-200 and H-600, respectively) for 48 hr. β-Actin was used as loading control. E. Caspase-9-deficient (Jurkat w/o C9) and caspase-9-reconstituted (Jurkat C9) Jurkat cells were treated without (control) or with 600 μM hydralazine (H-600) for 48 hr. The inset figure shows the levels of caspase-9 and β-actin in caspase-9-reconstituted and -lacking cells, as determined by Western-blot. A nonspecific protein was detected by the caspase-3 antibody (*ns). In A, C and E, sub-G1 apoptotic cells were analyzed by flow cytometry. Error bars show SEM from three independent experiments. *p

    Journal: Oncotarget

    Article Title: The antihypertensive drug hydralazine activates the intrinsic pathway of apoptosis and causes DNA damage in leukemic T cells

    doi: 10.18632/oncotarget.7871

    Figure Lengend Snippet: Induction of caspase-dependent apoptosis by hydralazine in leukemic T cells A. Jurkat, CEM-6, MOLT-4 and peripheral blood lymphocytes (PBL) were treated with different doses of hydralazine (40, 80, 200, 400 and 600 μM; upper panel) or procainamide (100, 250 and 500 μM; lower panel) for either 48 hr (T cell lines) or 72 hr (PBL in upper panel). B. Expression of DNTM1 was determined by Western blotting in Jurkat cells after treatment for 24 and 48 hr without (C) or with the indicated concentrations of hydralazine and procainamide. C. Jurkat cells were preincubated for 1 hr in the absence or in the presence of the caspase inhibitor Q-VD-OPh (20 μM) before treatment without (control) or with 200 and 600 μM hydralazine (H-200 and H-600, respectively) for 24 and 48 hr. D. Activation of caspases-8, -3 and -9 was determined by Western blot in Jurkat cells treated without (C) or with 80, 200 and 600 μM hydralazine (H-80, H-200 and H-600, respectively) for 48 hr. β-Actin was used as loading control. E. Caspase-9-deficient (Jurkat w/o C9) and caspase-9-reconstituted (Jurkat C9) Jurkat cells were treated without (control) or with 600 μM hydralazine (H-600) for 48 hr. The inset figure shows the levels of caspase-9 and β-actin in caspase-9-reconstituted and -lacking cells, as determined by Western-blot. A nonspecific protein was detected by the caspase-3 antibody (*ns). In A, C and E, sub-G1 apoptotic cells were analyzed by flow cytometry. Error bars show SEM from three independent experiments. *p

    Article Snippet: Q-VD-OPh, a wide-spectrum caspase inhibitor, and anti-human caspase-9 monoclonal antibody were from R & D Systems (Minneapolis, MN).

    Techniques: Expressing, Western Blot, Activation Assay, Flow Cytometry, Cytometry

    Induction of mitochondrial events by hydralazine in caspase-9 deficient cells A. Bak activation was analyzed by flow cytometry in caspase-9-deficient (Jurkat w/o C9) and caspase-9-reconstituted (Jurkat C9) Jurkat cells after treatment without (control) or with 400 and 600 μM hydralazine (H-400 and H-600, respectively) for 24 hr. B. Mitochondrial membrane potential and ROS production were determined by flow cytometry in caspase-9-deficient (Jurkat w/o C9) and caspase-9-reconstituted (Jurkat C9) Jurkat cells treated without (control) or with 600 μM hydralazine for 24 hr.

    Journal: Oncotarget

    Article Title: The antihypertensive drug hydralazine activates the intrinsic pathway of apoptosis and causes DNA damage in leukemic T cells

    doi: 10.18632/oncotarget.7871

    Figure Lengend Snippet: Induction of mitochondrial events by hydralazine in caspase-9 deficient cells A. Bak activation was analyzed by flow cytometry in caspase-9-deficient (Jurkat w/o C9) and caspase-9-reconstituted (Jurkat C9) Jurkat cells after treatment without (control) or with 400 and 600 μM hydralazine (H-400 and H-600, respectively) for 24 hr. B. Mitochondrial membrane potential and ROS production were determined by flow cytometry in caspase-9-deficient (Jurkat w/o C9) and caspase-9-reconstituted (Jurkat C9) Jurkat cells treated without (control) or with 600 μM hydralazine for 24 hr.

    Article Snippet: Q-VD-OPh, a wide-spectrum caspase inhibitor, and anti-human caspase-9 monoclonal antibody were from R & D Systems (Minneapolis, MN).

    Techniques: Activation Assay, Flow Cytometry, Cytometry

    Inhibition of SHH triggers cancer cell death via CDON-induced apoptosis. (A) Quantification of SHH expression by Q-RT-PCR in a panel of 45 human colorectal tumors and paired normal tissues. Data are presented as a ratio of SHH expression between tumor and normal tissue for each sample. Overexpression of SHH is considered when more than a 1.5-fold increase in the tumor is observed (indicated by the dashed line). (B) Quantification of SHH expression by Q-RT-PCR in a panel of 105 human tumors. Each tumor sample was compared to a normal paired tissue. For each type of tissue, the percentage of tumors showing an increase in SHH (considered when a more than 1.5-fold increase in expression is observed as compared to the normal tissue) is indicated. (C) Quantification of endogenous secreted SHH by ELISA assay in A549, H522, and H460 cells culture medium. H460 cell line is presented as a negative control. (D) Quantification of endogenous secreted SHH by ELISA assay in the culture medium of A549 cells transfected with scramble or SHH siRNA. (E) CDON immunofluorescence staining of A549 cells transfected with scramble or CDON siRNA using a CDON-specific antibody (in green) as described in the methods section. Nuclei were stained with Hoechst (in blue). (F) Apoptotic cell death induction as measured by TUNEL staining was quantified in A549 and H522 cells transfected with SHH siRNA alone or together with CDON siRNA. (G) Caspase-9 activity was measured in A549 cells 18 h after transfection with SHH siRNA alone or together with CDON siRNA. (H) Nude mice were engrafted with A549 cells by subcutaneous injection of 10 million cells. When the mean tumor volume reached approximately 100 mm 3 , animals were treated twice a week by i.p. injection of scramble or SHH siRNA alone or in combination with CDON siRNA during 4 wk. Mean tumor volume and number of animals for each group are indicated. (I) Representative images of scr siRNA, SHH siRNA, or SHH siRNA+CDON siRNA-treated tumors on day 35. (J) Apoptosis quantification by caspase-3 activity assay on xenografted tumor lysates analyzed after 1 wk of treatment with siRNAs. For (H), error bars indicate s.e.m. Statistical treatment of the data was performed using a two-sided Mann–Whitney test compared to scramble siRNA-treated condition (* p

    Journal: PLoS Biology

    Article Title: Sonic Hedgehog Promotes Tumor Cell Survival by Inhibiting CDON Pro-Apoptotic Activity

    doi: 10.1371/journal.pbio.1001623

    Figure Lengend Snippet: Inhibition of SHH triggers cancer cell death via CDON-induced apoptosis. (A) Quantification of SHH expression by Q-RT-PCR in a panel of 45 human colorectal tumors and paired normal tissues. Data are presented as a ratio of SHH expression between tumor and normal tissue for each sample. Overexpression of SHH is considered when more than a 1.5-fold increase in the tumor is observed (indicated by the dashed line). (B) Quantification of SHH expression by Q-RT-PCR in a panel of 105 human tumors. Each tumor sample was compared to a normal paired tissue. For each type of tissue, the percentage of tumors showing an increase in SHH (considered when a more than 1.5-fold increase in expression is observed as compared to the normal tissue) is indicated. (C) Quantification of endogenous secreted SHH by ELISA assay in A549, H522, and H460 cells culture medium. H460 cell line is presented as a negative control. (D) Quantification of endogenous secreted SHH by ELISA assay in the culture medium of A549 cells transfected with scramble or SHH siRNA. (E) CDON immunofluorescence staining of A549 cells transfected with scramble or CDON siRNA using a CDON-specific antibody (in green) as described in the methods section. Nuclei were stained with Hoechst (in blue). (F) Apoptotic cell death induction as measured by TUNEL staining was quantified in A549 and H522 cells transfected with SHH siRNA alone or together with CDON siRNA. (G) Caspase-9 activity was measured in A549 cells 18 h after transfection with SHH siRNA alone or together with CDON siRNA. (H) Nude mice were engrafted with A549 cells by subcutaneous injection of 10 million cells. When the mean tumor volume reached approximately 100 mm 3 , animals were treated twice a week by i.p. injection of scramble or SHH siRNA alone or in combination with CDON siRNA during 4 wk. Mean tumor volume and number of animals for each group are indicated. (I) Representative images of scr siRNA, SHH siRNA, or SHH siRNA+CDON siRNA-treated tumors on day 35. (J) Apoptosis quantification by caspase-3 activity assay on xenografted tumor lysates analyzed after 1 wk of treatment with siRNAs. For (H), error bars indicate s.e.m. Statistical treatment of the data was performed using a two-sided Mann–Whitney test compared to scramble siRNA-treated condition (* p

    Article Snippet: Co-immunprecipitations and immunoblots were performed as already described using anti-CDON (1/2,000, R & D Systems), anti-FlagM2 (1/5,000, Sigma), anti-HA (1∶7,500, Sigma), anti-caspase-9 (1∶1,000, Cell Signaling), or anti-β-actin (1∶1,000, Chemicon) primary antibodies.

    Techniques: Inhibition, Expressing, Reverse Transcription Polymerase Chain Reaction, Over Expression, Enzyme-linked Immunosorbent Assay, Negative Control, Transfection, Immunofluorescence, Staining, TUNEL Assay, Activity Assay, Mouse Assay, Injection, Caspase-3 Activity Assay, MANN-WHITNEY

    CDON triggers apoptosis through CDON proteolytic cleavage and recruitment and activation of caspase-9. (A) In vitro –translated CDON intracellular domain (CDON-IC) wild-type (wt) or mutated on one (left panel) or two (right panel) aspartic acid residues were incubated in the absence or in the presence of recombinant purified active caspase-3. Autoradiographs show the cleavage by caspase-3 of CDON-IC wt, whereas CDON-IC–D1153N was weakly cleaved and CDON-IC–D1153N–D1164N was almost completely resistant to cleavage. The band appearing in the CDON D1164N–D1153N probably represents a cryptic site. (B) Schematic representation of CDON and its different mutant constructs. CDON-main (D1153) and secondary (D1178) caspase cleavage sites are shown. (C–D) Apoptotic cell death induction as measured by caspase-3 activity (C) was quantified in HEK293T cells transfected with wild-type (CDON) full-length or mutated full-length CDON expression plasmids and by TUNEL (D) staining was quantified in HEK293T cells transfected with wild-type (CDON) full-length or mutated full-length CDON expression plasmids. Quantifications of TUNEL positive cells (orange) and nuclei (Hoechst in blue) are shown. (E) Apoptotic cell death induction as measured by caspase-3 activity was quantified in HEK293T cells transfected with constructs encoding full-length CDON or the CDON hypothetical fragments resulting from its cleavage by caspase at D1153 (CDON1–1153 and CDON 1154–1250). (F) HEK293T cells were transfected with constructs encoding CDON and/or caspase-9 and cell lysates were subjected to immunoprecipitation with a CDON-specific antibody. CDON and caspase-9 proteins were detected by Western blot in immunoprecipitated and input fractions. (G) Caspase-9 activity was quantified in HEK293T cells transfected with wild-type (CDON) full-length or mutated full-length CDON (D1153N) expression plasmids. For (C–F) and (G), data are means of at least three independent assays. Error bars indicate s.d.

    Journal: PLoS Biology

    Article Title: Sonic Hedgehog Promotes Tumor Cell Survival by Inhibiting CDON Pro-Apoptotic Activity

    doi: 10.1371/journal.pbio.1001623

    Figure Lengend Snippet: CDON triggers apoptosis through CDON proteolytic cleavage and recruitment and activation of caspase-9. (A) In vitro –translated CDON intracellular domain (CDON-IC) wild-type (wt) or mutated on one (left panel) or two (right panel) aspartic acid residues were incubated in the absence or in the presence of recombinant purified active caspase-3. Autoradiographs show the cleavage by caspase-3 of CDON-IC wt, whereas CDON-IC–D1153N was weakly cleaved and CDON-IC–D1153N–D1164N was almost completely resistant to cleavage. The band appearing in the CDON D1164N–D1153N probably represents a cryptic site. (B) Schematic representation of CDON and its different mutant constructs. CDON-main (D1153) and secondary (D1178) caspase cleavage sites are shown. (C–D) Apoptotic cell death induction as measured by caspase-3 activity (C) was quantified in HEK293T cells transfected with wild-type (CDON) full-length or mutated full-length CDON expression plasmids and by TUNEL (D) staining was quantified in HEK293T cells transfected with wild-type (CDON) full-length or mutated full-length CDON expression plasmids. Quantifications of TUNEL positive cells (orange) and nuclei (Hoechst in blue) are shown. (E) Apoptotic cell death induction as measured by caspase-3 activity was quantified in HEK293T cells transfected with constructs encoding full-length CDON or the CDON hypothetical fragments resulting from its cleavage by caspase at D1153 (CDON1–1153 and CDON 1154–1250). (F) HEK293T cells were transfected with constructs encoding CDON and/or caspase-9 and cell lysates were subjected to immunoprecipitation with a CDON-specific antibody. CDON and caspase-9 proteins were detected by Western blot in immunoprecipitated and input fractions. (G) Caspase-9 activity was quantified in HEK293T cells transfected with wild-type (CDON) full-length or mutated full-length CDON (D1153N) expression plasmids. For (C–F) and (G), data are means of at least three independent assays. Error bars indicate s.d.

    Article Snippet: Co-immunprecipitations and immunoblots were performed as already described using anti-CDON (1/2,000, R & D Systems), anti-FlagM2 (1/5,000, Sigma), anti-HA (1∶7,500, Sigma), anti-caspase-9 (1∶1,000, Cell Signaling), or anti-β-actin (1∶1,000, Chemicon) primary antibodies.

    Techniques: Activation Assay, In Vitro, Incubation, Recombinant, Purification, Mutagenesis, Construct, Activity Assay, Transfection, Expressing, TUNEL Assay, Staining, Immunoprecipitation, Western Blot

    API5 did not interact with caspase‐9 and caspase‐1 ) 24 h post‐treatment. The total lysates and the immunoprecipitated‐eluted samples were analysed by Western blot. 293T cells were transfected with pCMV2‐Flag‐Caspase‐1 or pCMV3‐Flag‐Caspase‐9 for 48 h. The Flag‐tagged caspases were precipitated by anti‐Flag M2 Magnetic Beads. The anti‐Flag M2 Magnetic Beads were washed 5 times and then incubated overnight at 4°C with recombinant API5. After washing (3×), the beads were re‐suspended in SDS buffer and subjected to Western blot analysis. Source data are available online for this figure.

    Journal: EMBO Reports

    Article Title: Apoptosis inhibitor 5 is an endogenous inhibitor of caspase‐2

    doi: 10.15252/embr.201643744

    Figure Lengend Snippet: API5 did not interact with caspase‐9 and caspase‐1 ) 24 h post‐treatment. The total lysates and the immunoprecipitated‐eluted samples were analysed by Western blot. 293T cells were transfected with pCMV2‐Flag‐Caspase‐1 or pCMV3‐Flag‐Caspase‐9 for 48 h. The Flag‐tagged caspases were precipitated by anti‐Flag M2 Magnetic Beads. The anti‐Flag M2 Magnetic Beads were washed 5 times and then incubated overnight at 4°C with recombinant API5. After washing (3×), the beads were re‐suspended in SDS buffer and subjected to Western blot analysis. Source data are available online for this figure.

    Article Snippet: The rest of the samples was mixed with 4.5 μl of caspase‐9 antibody (Cell Signalling) and incubated overnight at 4°C in a vertical rotator.

    Techniques: Immunoprecipitation, Western Blot, Transfection, Magnetic Beads, Incubation, Recombinant

    M1 inhibits Hsp70-mediated retardation of caspase activation in a cell-free system. ( a and b ) Cytosolic extracts prepared from Jurkat T cells were incubated with cytochrome c (10 μ M) and dATP (1 mM) in the presence or absence of recombinant human Hsp70 (3.58 μ M) and or recombinant M1 (10 μ g/ml) for the indicated time periods. Cleavage activities of caspase-9 and caspase-3 were determined by fluorometric quantification of LEHD-AMC and DEVD-AFC substrate cleavage, respectively. Results are representative of three independent experiments. Values represent means±S.D. of one experiment with three measurements taken. ( c ) Caspase cleavage was assessed by immunoblot analysis after exogenous addition of cytochrome c (10 μ M) and dATP (1 mM) in presence or absence of recombinant Hsp70 (3.58 μ M) and recombinant M1(10 μ g/ml)

    Journal: Cell Death & Disease

    Article Title: Cell death regulation during influenza A virus infection by matrix (M1) protein: a model of viral control over the cellular survival pathway

    doi: 10.1038/cddis.2011.75

    Figure Lengend Snippet: M1 inhibits Hsp70-mediated retardation of caspase activation in a cell-free system. ( a and b ) Cytosolic extracts prepared from Jurkat T cells were incubated with cytochrome c (10 μ M) and dATP (1 mM) in the presence or absence of recombinant human Hsp70 (3.58 μ M) and or recombinant M1 (10 μ g/ml) for the indicated time periods. Cleavage activities of caspase-9 and caspase-3 were determined by fluorometric quantification of LEHD-AMC and DEVD-AFC substrate cleavage, respectively. Results are representative of three independent experiments. Values represent means±S.D. of one experiment with three measurements taken. ( c ) Caspase cleavage was assessed by immunoblot analysis after exogenous addition of cytochrome c (10 μ M) and dATP (1 mM) in presence or absence of recombinant Hsp70 (3.58 μ M) and recombinant M1(10 μ g/ml)

    Article Snippet: Antibodies against caspase-9 (9501, 9502), caspase-7 (9491, 9491), caspase-3 (9662, 9664), PARP (9541, 9542), Bid (2002) and Flag (DYKDDDDK, 2368) were from Cell Signaling Technology, Inc (Danvers, MA, USA).

    Techniques: Activation Assay, Incubation, Recombinant

    M1-mediated early activation of caspases in staurosporine induced 293T cells. ( a and b ) 293T cells were transiently transfected with empty control vector or pcD-M1 and left untreated for 36 h before control and transfected cells were treated with staurosporine (1 μ M, 6 h) for the indicated times. Actual enzymatic activity of caspase-9 and caspase-3 in cell lysates of control 293T cells or M1-expressing 293T cells treated with staurosporine was measured fluorometrically by cleavage of LEHD-AMC and DEVD-AFC, respectively, for the given times. Results are representative of three independent experiments. Values represent means±S.D. of one experiment with three measurements taken. ( c ) Immunoblotting was performed using whole-cell lysates for cleavage of caspase-9, caspase-7, caspase-3 and PARP. β -Actin served as an internal loading control

    Journal: Cell Death & Disease

    Article Title: Cell death regulation during influenza A virus infection by matrix (M1) protein: a model of viral control over the cellular survival pathway

    doi: 10.1038/cddis.2011.75

    Figure Lengend Snippet: M1-mediated early activation of caspases in staurosporine induced 293T cells. ( a and b ) 293T cells were transiently transfected with empty control vector or pcD-M1 and left untreated for 36 h before control and transfected cells were treated with staurosporine (1 μ M, 6 h) for the indicated times. Actual enzymatic activity of caspase-9 and caspase-3 in cell lysates of control 293T cells or M1-expressing 293T cells treated with staurosporine was measured fluorometrically by cleavage of LEHD-AMC and DEVD-AFC, respectively, for the given times. Results are representative of three independent experiments. Values represent means±S.D. of one experiment with three measurements taken. ( c ) Immunoblotting was performed using whole-cell lysates for cleavage of caspase-9, caspase-7, caspase-3 and PARP. β -Actin served as an internal loading control

    Article Snippet: Antibodies against caspase-9 (9501, 9502), caspase-7 (9491, 9491), caspase-3 (9662, 9664), PARP (9541, 9542), Bid (2002) and Flag (DYKDDDDK, 2368) were from Cell Signaling Technology, Inc (Danvers, MA, USA).

    Techniques: Activation Assay, Transfection, Plasmid Preparation, Activity Assay, Expressing

    Amino acid 128–165 is responsible for direct binding with Hsp70 to prevent Apaf-1 binding and to aid in caspase-9 processing induced by cytochrome c /ATP. ( a ) His-tagged M1or M1Δ128–165 (10 μ g) was immobilized on Ni +2 overnight in HBS at 4 °C, and then washed and incubated with recombinant Hsp70 (10 μ g) for 4 h at 4 °C. Associated Hsp70 was detected by immunoblotting. Wild-type Hsp70 associated with native M1 but not with M1Δ128–165. ( b ) Jurkat cell extracts were Hsp70 immunodepleted, before recombinant Hsp70 and M1 or M1Δ128–165 proteins were added to it incubated and Apaf-1 was immunoprecipitated. Hsp70 did not associate with Apaf-1 when native M1 was present, but did associate in the absence of M1 or in the presence of M1Δ128–165 protein. ( c ) Co-addition of recombinant M1 enhances processing of procaspase-9 induced by dATP or ATP and cytochrome c in Jurkat cell extracts in the presence of wild-type Hsp70, whereas in the presence of M1Δ128–165 protein, caspase-9 activation is inhibited

    Journal: Cell Death & Disease

    Article Title: Cell death regulation during influenza A virus infection by matrix (M1) protein: a model of viral control over the cellular survival pathway

    doi: 10.1038/cddis.2011.75

    Figure Lengend Snippet: Amino acid 128–165 is responsible for direct binding with Hsp70 to prevent Apaf-1 binding and to aid in caspase-9 processing induced by cytochrome c /ATP. ( a ) His-tagged M1or M1Δ128–165 (10 μ g) was immobilized on Ni +2 overnight in HBS at 4 °C, and then washed and incubated with recombinant Hsp70 (10 μ g) for 4 h at 4 °C. Associated Hsp70 was detected by immunoblotting. Wild-type Hsp70 associated with native M1 but not with M1Δ128–165. ( b ) Jurkat cell extracts were Hsp70 immunodepleted, before recombinant Hsp70 and M1 or M1Δ128–165 proteins were added to it incubated and Apaf-1 was immunoprecipitated. Hsp70 did not associate with Apaf-1 when native M1 was present, but did associate in the absence of M1 or in the presence of M1Δ128–165 protein. ( c ) Co-addition of recombinant M1 enhances processing of procaspase-9 induced by dATP or ATP and cytochrome c in Jurkat cell extracts in the presence of wild-type Hsp70, whereas in the presence of M1Δ128–165 protein, caspase-9 activation is inhibited

    Article Snippet: Antibodies against caspase-9 (9501, 9502), caspase-7 (9491, 9491), caspase-3 (9662, 9664), PARP (9541, 9542), Bid (2002) and Flag (DYKDDDDK, 2368) were from Cell Signaling Technology, Inc (Danvers, MA, USA).

    Techniques: Binding Assay, Incubation, Recombinant, Immunoprecipitation, Activation Assay

    Involvement of M1 in mitochondria-mediated caspase activation during influenza A/PR8 infection. ( a and b ) A549 cells were transfected with control siRNA or M1 siRNA (60 nmol), and 24 h later, were infected with 1 m.o.i. (multiplicity of infection) of influenza A/PR8 virus. Caspase-9 and caspase-3 activities were determined by hydrolysis of the LEHD-AMC and DEVD-AFC substrates, respectively. Results are representative of three independent experiments. Values represent means±S.D. of one experiment with three measurements taken. ( c ) Caspase processing was assayed by immunoblot analysis for the indicated times. Zymogens and cleavage products are indicated. ( d ) Expression of M1 was assessed by immunoblotting in PR8-infected cells and M1-siRNA-treated PR8-infected cells for the indicated times

    Journal: Cell Death & Disease

    Article Title: Cell death regulation during influenza A virus infection by matrix (M1) protein: a model of viral control over the cellular survival pathway

    doi: 10.1038/cddis.2011.75

    Figure Lengend Snippet: Involvement of M1 in mitochondria-mediated caspase activation during influenza A/PR8 infection. ( a and b ) A549 cells were transfected with control siRNA or M1 siRNA (60 nmol), and 24 h later, were infected with 1 m.o.i. (multiplicity of infection) of influenza A/PR8 virus. Caspase-9 and caspase-3 activities were determined by hydrolysis of the LEHD-AMC and DEVD-AFC substrates, respectively. Results are representative of three independent experiments. Values represent means±S.D. of one experiment with three measurements taken. ( c ) Caspase processing was assayed by immunoblot analysis for the indicated times. Zymogens and cleavage products are indicated. ( d ) Expression of M1 was assessed by immunoblotting in PR8-infected cells and M1-siRNA-treated PR8-infected cells for the indicated times

    Article Snippet: Antibodies against caspase-9 (9501, 9502), caspase-7 (9491, 9491), caspase-3 (9662, 9664), PARP (9541, 9542), Bid (2002) and Flag (DYKDDDDK, 2368) were from Cell Signaling Technology, Inc (Danvers, MA, USA).

    Techniques: Activation Assay, Infection, Transfection, Expressing

    Azathioprine induces a mitochondrial pathway of apoptosis. ( a ) Activity of caspase-3, -8, and -9 upon treatment of T cells with 6-MP. CD45RA and CD45RO T cell subsets were stimulated with antibodies to CD3 and CD28 and recombinant IL-2 for 5 days in the presence or absence of 6-MP, as indicated. There was a marked induction of caspase-9 activity upon azathioprine treatment. A second independent experiment showed similar results (data not shown). Data on caspase-9 activity from three independent healthy blood donors are shown in the right lower panel. ( b ) Specific blockade of caspase-9 by acetyl-LEHD-CHO (Ac-LEHD-CHO) suppresses 6-MP–induced apoptosis. CD4 + T lymphocytes from the peripheral blood of healthy volunteers were stimulated with antibodies to CD3 and CD28 in the presence or absence of 6-MP, 10 μM acetyl-LEHD-CHO, and 10 μm acetyl-IETD-CHO. Although acetyl-IETD-CHO had little effect, 6-MP–induced T cell apoptosis could be suppressed by acetyl-LEHD-CHO. ( c ) Measurement of ΔΨ m in primary CD4 + T lymphocytes upon treatment with azathioprine, 6-MP, and FCCP (positive control). Peripheral blood CD4 + T cells from healthy volunteers were stimulated with antibodies to CD3 and CD28 and recombinant IL-2 and cultured in the presence or absence of azathioprine or 6-MP for 5 days as indicated. Cells were then loaded with JC-1 for 20 minutes followed by FACS analysis to determine ΔΨ m . Both azathioprine and 6-MP as well as FCCP led to a marked reduction of ΔΨ m as compared with untreated primary CD4 + T cells. One representative experiment of two is shown. Aza, azathioprine; UT, untreated.

    Journal: Journal of Clinical Investigation

    Article Title: CD28-dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes

    doi: 10.1172/JCI200316432

    Figure Lengend Snippet: Azathioprine induces a mitochondrial pathway of apoptosis. ( a ) Activity of caspase-3, -8, and -9 upon treatment of T cells with 6-MP. CD45RA and CD45RO T cell subsets were stimulated with antibodies to CD3 and CD28 and recombinant IL-2 for 5 days in the presence or absence of 6-MP, as indicated. There was a marked induction of caspase-9 activity upon azathioprine treatment. A second independent experiment showed similar results (data not shown). Data on caspase-9 activity from three independent healthy blood donors are shown in the right lower panel. ( b ) Specific blockade of caspase-9 by acetyl-LEHD-CHO (Ac-LEHD-CHO) suppresses 6-MP–induced apoptosis. CD4 + T lymphocytes from the peripheral blood of healthy volunteers were stimulated with antibodies to CD3 and CD28 in the presence or absence of 6-MP, 10 μM acetyl-LEHD-CHO, and 10 μm acetyl-IETD-CHO. Although acetyl-IETD-CHO had little effect, 6-MP–induced T cell apoptosis could be suppressed by acetyl-LEHD-CHO. ( c ) Measurement of ΔΨ m in primary CD4 + T lymphocytes upon treatment with azathioprine, 6-MP, and FCCP (positive control). Peripheral blood CD4 + T cells from healthy volunteers were stimulated with antibodies to CD3 and CD28 and recombinant IL-2 and cultured in the presence or absence of azathioprine or 6-MP for 5 days as indicated. Cells were then loaded with JC-1 for 20 minutes followed by FACS analysis to determine ΔΨ m . Both azathioprine and 6-MP as well as FCCP led to a marked reduction of ΔΨ m as compared with untreated primary CD4 + T cells. One representative experiment of two is shown. Aza, azathioprine; UT, untreated.

    Article Snippet: Since these data suggested that 6-MP–induced apoptosis might involve activation of a mitochondrial pathway of apoptosis involving caspase-9, we focused in consecutive studies on the role of caspase-9 in 6-MP–induced apoptosis.

    Techniques: Activity Assay, Recombinant, Positive Control, Cell Culture, FACS

    Bcl-X L inhibits the association of Apaf-1 with caspase-9. 293T cells were transfected with indicated plasmids and the lysates immunoprecipitated with anti-HA antibody. ( Upper ) Western blot analysis of immunoprecipitated caspase-9 and coimmunoprecipitated Apaf-1. ( Lower ) Western blot analysis of total lysates with anti-Myc and anti-Flag antibody.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Bcl-XL interacts with Apaf-1 and inhibits Apaf-1-dependent caspase-9 activation

    doi:

    Figure Lengend Snippet: Bcl-X L inhibits the association of Apaf-1 with caspase-9. 293T cells were transfected with indicated plasmids and the lysates immunoprecipitated with anti-HA antibody. ( Upper ) Western blot analysis of immunoprecipitated caspase-9 and coimmunoprecipitated Apaf-1. ( Lower ) Western blot analysis of total lysates with anti-Myc and anti-Flag antibody.

    Article Snippet: Caspase-9 was translated in vitro from a pcDNA-3-caspase-9 plasmid in the presence of [35 S]methionine (Amersham) by using a Promega TNT transcription/translation kit.

    Techniques: Transfection, Immunoprecipitation, Western Blot

    Regulation of caspase-9 by full-length and N-terminal Apaf-1. ( A ) Apaf-1 enhances caspase-9-induced apoptosis. 293T cells were transiently transfected with a reporter pcDNA3-β-galactosidase plus the indicated plasmids. WT, full-length Apaf-1-Myc; N, N-terminal Apaf-1-Myc; C, C-terminal Apaf-1-Myc; UT, Untagged full-length Apaf-1. The results represent the percentage of blue cells that exhibit morphologic features of apoptosis and are given as the mean +/− SD of triplicate cultures. ( B ) Apaf-1 promotes caspase-9 activation in vivo . Lysates from cells transfected with the indicated plasmids were immunoblotted with anti-caspase-9 ( Upper ) or anti-PARP antibodies ( Lower ). Arrows indicate full-length and processed caspase-9 and PARP fragments. Asterisk indicates nonspecific band.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Bcl-XL interacts with Apaf-1 and inhibits Apaf-1-dependent caspase-9 activation

    doi:

    Figure Lengend Snippet: Regulation of caspase-9 by full-length and N-terminal Apaf-1. ( A ) Apaf-1 enhances caspase-9-induced apoptosis. 293T cells were transiently transfected with a reporter pcDNA3-β-galactosidase plus the indicated plasmids. WT, full-length Apaf-1-Myc; N, N-terminal Apaf-1-Myc; C, C-terminal Apaf-1-Myc; UT, Untagged full-length Apaf-1. The results represent the percentage of blue cells that exhibit morphologic features of apoptosis and are given as the mean +/− SD of triplicate cultures. ( B ) Apaf-1 promotes caspase-9 activation in vivo . Lysates from cells transfected with the indicated plasmids were immunoblotted with anti-caspase-9 ( Upper ) or anti-PARP antibodies ( Lower ). Arrows indicate full-length and processed caspase-9 and PARP fragments. Asterisk indicates nonspecific band.

    Article Snippet: Caspase-9 was translated in vitro from a pcDNA-3-caspase-9 plasmid in the presence of [35 S]methionine (Amersham) by using a Promega TNT transcription/translation kit.

    Techniques: Transfection, Activation Assay, In Vivo

    Bcl-X L interacts with caspase-9 and Apaf-1. ( A ) Bcl-X L interacts with caspase-9. ( Upper ) Western blot analysis of immunoprecipitated Bcl-X L and coimmunoprecipitated pro-caspase-9 and processed form (p35). ( Lower ) The expression of caspase-9 in total lysate. ( B and C ) Bcl-X L interacts with Apaf-1. 293T cells were transfected with indicated plasmids and the lysates immunoprecipitated with anti-Flag ( B ) or anti-Myc ( C ) antibody. WT, full-length Apaf-1-Myc; N, N-terminal Apaf-1-Myc; C, C-terminal Apaf-1-Myc. Panels show Western blot analysis of coimmunoprecipitated Apaf-1 and Bcl-X L proteins. Asterisk indicates nonspecific band.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Bcl-XL interacts with Apaf-1 and inhibits Apaf-1-dependent caspase-9 activation

    doi:

    Figure Lengend Snippet: Bcl-X L interacts with caspase-9 and Apaf-1. ( A ) Bcl-X L interacts with caspase-9. ( Upper ) Western blot analysis of immunoprecipitated Bcl-X L and coimmunoprecipitated pro-caspase-9 and processed form (p35). ( Lower ) The expression of caspase-9 in total lysate. ( B and C ) Bcl-X L interacts with Apaf-1. 293T cells were transfected with indicated plasmids and the lysates immunoprecipitated with anti-Flag ( B ) or anti-Myc ( C ) antibody. WT, full-length Apaf-1-Myc; N, N-terminal Apaf-1-Myc; C, C-terminal Apaf-1-Myc. Panels show Western blot analysis of coimmunoprecipitated Apaf-1 and Bcl-X L proteins. Asterisk indicates nonspecific band.

    Article Snippet: Caspase-9 was translated in vitro from a pcDNA-3-caspase-9 plasmid in the presence of [35 S]methionine (Amersham) by using a Promega TNT transcription/translation kit.

    Techniques: Western Blot, Immunoprecipitation, Expressing, Transfection

    Purified Bcl-X L inhibits Apaf-1-dependent maturation of caspase-9 in vitro . ( A ) Apaf-1-dependent processing of caspase-9 by Apaf-1 in vitro . In the last two lanes normal rabbit serum (control) or anti-Apaf-1 serum were added 30 min prior to the reaction. ( B ) N-terminal Apaf-1 mutant (residues 1–559) activates caspase-9 independently of cytochrome c and dATP. Extracts from 293T cells transfected with pcDNA3 (control extract) or pcDNA3-N-Apaf-1 (N-Apaf-1 extract) were used in the analysis. ( C ) Regulation of caspase-9 maturation by recombinant Bcl-X L . Cellular extracts were incubated with indicated amounts of recombinant Bcl-X L or Bad and then cytochrome c and dATP were added to the reaction. ( Right ) Extracts from cells expressing a N-terminal Apaf-1 mutant (residues 1–559) were incubated with indicated amounts of recombinant Bcl-X L . Proform and processed caspase-9 are indicated by arrows.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Bcl-XL interacts with Apaf-1 and inhibits Apaf-1-dependent caspase-9 activation

    doi:

    Figure Lengend Snippet: Purified Bcl-X L inhibits Apaf-1-dependent maturation of caspase-9 in vitro . ( A ) Apaf-1-dependent processing of caspase-9 by Apaf-1 in vitro . In the last two lanes normal rabbit serum (control) or anti-Apaf-1 serum were added 30 min prior to the reaction. ( B ) N-terminal Apaf-1 mutant (residues 1–559) activates caspase-9 independently of cytochrome c and dATP. Extracts from 293T cells transfected with pcDNA3 (control extract) or pcDNA3-N-Apaf-1 (N-Apaf-1 extract) were used in the analysis. ( C ) Regulation of caspase-9 maturation by recombinant Bcl-X L . Cellular extracts were incubated with indicated amounts of recombinant Bcl-X L or Bad and then cytochrome c and dATP were added to the reaction. ( Right ) Extracts from cells expressing a N-terminal Apaf-1 mutant (residues 1–559) were incubated with indicated amounts of recombinant Bcl-X L . Proform and processed caspase-9 are indicated by arrows.

    Article Snippet: Caspase-9 was translated in vitro from a pcDNA-3-caspase-9 plasmid in the presence of [35 S]methionine (Amersham) by using a Promega TNT transcription/translation kit.

    Techniques: Purification, In Vitro, Mutagenesis, Transfection, Recombinant, Incubation, Expressing

    Apaf-1 interacts with several caspases with long prodomains in mammalian cells. ( A ) Schematic drawing of the Apaf-1 constructs used in this study. ( B–D ) Interaction of Apaf-1 with caspase-4, caspase-9, and CED-3. 293T cells were cotransfected with the indicated tagged caspases and control vector, or tagged Apaf-1, N-terminal, or C-terminal Apaf-1 constructs (Apaf-1-Myc, N-Apaf-1-Myc, and C-Apaf-1-Myc, respectively). ( Upper ) Western blot analysis of immunoprecipitated protein complexes with indicated antibodies. ( Lower ) The expression of caspase-3 and -4 ( B ), caspase-9 ( C ), and CED-3 and Apaf-1 proteins ( D ). Reduced level of pro-caspase-9 in lane 3 of C is due to efficient N-Apaf-1-mediated processing of pro-caspase-9. IP: immunoprecipitation, WB: Western blot analysis. Asterisks indicate nonspecific bands.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Bcl-XL interacts with Apaf-1 and inhibits Apaf-1-dependent caspase-9 activation

    doi:

    Figure Lengend Snippet: Apaf-1 interacts with several caspases with long prodomains in mammalian cells. ( A ) Schematic drawing of the Apaf-1 constructs used in this study. ( B–D ) Interaction of Apaf-1 with caspase-4, caspase-9, and CED-3. 293T cells were cotransfected with the indicated tagged caspases and control vector, or tagged Apaf-1, N-terminal, or C-terminal Apaf-1 constructs (Apaf-1-Myc, N-Apaf-1-Myc, and C-Apaf-1-Myc, respectively). ( Upper ) Western blot analysis of immunoprecipitated protein complexes with indicated antibodies. ( Lower ) The expression of caspase-3 and -4 ( B ), caspase-9 ( C ), and CED-3 and Apaf-1 proteins ( D ). Reduced level of pro-caspase-9 in lane 3 of C is due to efficient N-Apaf-1-mediated processing of pro-caspase-9. IP: immunoprecipitation, WB: Western blot analysis. Asterisks indicate nonspecific bands.

    Article Snippet: Caspase-9 was translated in vitro from a pcDNA-3-caspase-9 plasmid in the presence of [35 S]methionine (Amersham) by using a Promega TNT transcription/translation kit.

    Techniques: Construct, Plasmid Preparation, Western Blot, Immunoprecipitation, Expressing

    Functional antagonism of Smac mimetics against XIAP linker-BIR2-BIR3 in a cell-free caspase-9 functional assay. Data shown in the figure are averages and standard deviations of duplicate wells in assay plates, and the figure is the representative of three

    Journal: Journal of medicinal chemistry

    Article Title: Potent Bivalent Smac Mimetics: Effect of the Linker on Binding to Inhibitor of Apoptosis Proteins (IAPs) and Anticancer Activity

    doi: 10.1021/jm101651b

    Figure Lengend Snippet: Functional antagonism of Smac mimetics against XIAP linker-BIR2-BIR3 in a cell-free caspase-9 functional assay. Data shown in the figure are averages and standard deviations of duplicate wells in assay plates, and the figure is the representative of three

    Article Snippet: For the caspase-9 activity assay, the enzymatic activity of active recombinant caspase-9 (Enzo Life Sciences) was evaluated by the caspase-Glo 9 Assay kit from Promega.

    Techniques: Functional Assay

    RGDS-survivin interaction. A: Bt-RGDS interaction with cell lysate: cytoplasmic extracts were immobilized onto nitrocellulose and incubated with bt-RGDS. Total binding in the presence of labeled RGDS and nonspecific binding in the presence of an excess of unlabeled RGDS are shown. Densitometry of three different experiments and one representative experiment are reported. B: RGDS interaction with intracellular proteins was assayed by co-precipitation. BSA (control), bt-RGES (specificity control) or bt-RGDS (1 mM) were incubated for 1 h at 4°C with streptavidin-coated dynabeads, then SK-MEL-110 lysate, pre-incubated for 4 h at 4°C with an excess of unlabeled RGDS, was added and incubated overnight at 4°C. Precipitated proteins were revealed by western blotting using various antibodies (anti pro-caspase-8, anti pro-caspase-9, anti pro-caspase-1, anti pro-caspase-3 and anti-survivin). Total lysate was used as positive control. This experiment was carried out three times. C: Purified recombinant proteins were spotted onto nitrocellulose (0.9 μg/spot). Membrane was incubated for 4 h at RT with bt-RGDS (1 mg/ml) (total binding) in the absence or in the presence of an excess of unlabeled RGDS (10 mg/ml) (nonspecific binding). Three independent experiments were performed.

    Journal: Molecular Cancer

    Article Title: Intracellular targets of RGDS peptide in melanoma cells

    doi: 10.1186/1476-4598-9-84

    Figure Lengend Snippet: RGDS-survivin interaction. A: Bt-RGDS interaction with cell lysate: cytoplasmic extracts were immobilized onto nitrocellulose and incubated with bt-RGDS. Total binding in the presence of labeled RGDS and nonspecific binding in the presence of an excess of unlabeled RGDS are shown. Densitometry of three different experiments and one representative experiment are reported. B: RGDS interaction with intracellular proteins was assayed by co-precipitation. BSA (control), bt-RGES (specificity control) or bt-RGDS (1 mM) were incubated for 1 h at 4°C with streptavidin-coated dynabeads, then SK-MEL-110 lysate, pre-incubated for 4 h at 4°C with an excess of unlabeled RGDS, was added and incubated overnight at 4°C. Precipitated proteins were revealed by western blotting using various antibodies (anti pro-caspase-8, anti pro-caspase-9, anti pro-caspase-1, anti pro-caspase-3 and anti-survivin). Total lysate was used as positive control. This experiment was carried out three times. C: Purified recombinant proteins were spotted onto nitrocellulose (0.9 μg/spot). Membrane was incubated for 4 h at RT with bt-RGDS (1 mg/ml) (total binding) in the absence or in the presence of an excess of unlabeled RGDS (10 mg/ml) (nonspecific binding). Three independent experiments were performed.

    Article Snippet: Samples were boiled and separated by SDS-PAGE as described above and incubated with antibody to survivin (1:200), caspase-3 (1:200), caspase-8 (1:200) (Santa Cruz Biotechnology, Alexa, CA), caspase-9 (1:1000) (Pharmingen, San Diego, CA), caspase-1 (1:500) (Alexis, San Diego, CA).

    Techniques: Incubation, Binding Assay, Labeling, Western Blot, Positive Control, Purification, Recombinant

    ILP-1 and ILP-2 inhibit processing of caspases 9 and 3 in a cell-free system. 293 cell extracts (1.5 mg) were incubated with recombinant, purified GST–ILP-1, GST–ILP-2 or control GST proteins (1 μg) for 30 min, activated with ATP for 30 min, and resolved by gel filtration chromatography on a Superose 6 column (Amersham Pharmacia) using an ÄKTA FPLC system (Amersham Pharmacia). Aliquots of the fractions eluted from the column were separated by SDS-PAGE, immunoblotted onto nitrocellulose, and probed with the indicated antibodies as follows. (A) Apaf-1 (Y. Lazebnik, Cold Spring Harbor Laboratories, N.Y.). The calculated molecular mass of Apaf-1 was consistent with the expected molecular mass of 130 kDa and is indicated (p130). (B) Caspase 9 (Upstate Biotechnology). The full-length (Pro) and processed 35- and 37-kDa species (p35/37) are indi- cated. (C) Caspase 3 (Pharmingen). The inactive precursor (Pro) and the 19- and 17-kDa processed, active forms (p19/17) are indicated. (D) Aliquots of the fractions were also evaluated for DEVDase activity, using DEVD-AFC (20 μM) as substrate as described in Materials and Methods. Molecular masses (in kilodaltons) are shown.

    Journal: Molecular and Cellular Biology

    Article Title: Molecular Cloning of ILP-2, a Novel Member of the Inhibitor of Apoptosis Protein Family †

    doi: 10.1128/MCB.21.13.4292-4301.2001

    Figure Lengend Snippet: ILP-1 and ILP-2 inhibit processing of caspases 9 and 3 in a cell-free system. 293 cell extracts (1.5 mg) were incubated with recombinant, purified GST–ILP-1, GST–ILP-2 or control GST proteins (1 μg) for 30 min, activated with ATP for 30 min, and resolved by gel filtration chromatography on a Superose 6 column (Amersham Pharmacia) using an ÄKTA FPLC system (Amersham Pharmacia). Aliquots of the fractions eluted from the column were separated by SDS-PAGE, immunoblotted onto nitrocellulose, and probed with the indicated antibodies as follows. (A) Apaf-1 (Y. Lazebnik, Cold Spring Harbor Laboratories, N.Y.). The calculated molecular mass of Apaf-1 was consistent with the expected molecular mass of 130 kDa and is indicated (p130). (B) Caspase 9 (Upstate Biotechnology). The full-length (Pro) and processed 35- and 37-kDa species (p35/37) are indi- cated. (C) Caspase 3 (Pharmingen). The inactive precursor (Pro) and the 19- and 17-kDa processed, active forms (p19/17) are indicated. (D) Aliquots of the fractions were also evaluated for DEVDase activity, using DEVD-AFC (20 μM) as substrate as described in Materials and Methods. Molecular masses (in kilodaltons) are shown.

    Article Snippet: After blocking the membranes in 5% dry milk in Tris-buffered saline with 0.2% Tween, the membranes were incubated with either anti-GST antibody (Santa Cruz Laboratories, Santa Cruz, Calif.), anti-Apaf-1 antibody (a kind gift of Y. Lazebnik, Cold Spring Harbor, N.Y.), or anti-caspase 9 antibodies (Pharmingen, San Diego, Calif. or Upstate Biotechnology, Lake Placid, N.Y.) as indicated in the figure legends and were visualized by enhanced chemiluminescence (ECL; Amersham Pharmacia).

    Techniques: Incubation, Recombinant, Purification, Filtration, Chromatography, Fast Protein Liquid Chromatography, SDS Page, Activity Assay

    ILP-2 inhibits apoptosis induced by Bax and caspase 9 plus Apaf-1 but not by Fas. Human embryonic kidney 293 cells were transfected with a GFP plasmid (0.5 μg/well) in combination with either a Bax expression plasmid (0.25 μg/well) (A), Fas expression plasmid (2 μg/well) (B), or Apaf-1 (2 μg/well) and caspase 9 (0.5 μg/well) plasmids, as indicated (C). Cells were cotransfected with a plasmid encoding GFP as a marker of transfection, together with a control plasmid or plasmids expressing ILP-1, ILP-2, or dominant-negative caspase 9 (C-9 DN) (2 μg/well) as indicated. Cells were fixed and stained with DAPI, and transfected wells were scored for viability based on nuclear morphology as described in Materials and Methods. Data shown are representative of at least three independent experiments.

    Journal: Molecular and Cellular Biology

    Article Title: Molecular Cloning of ILP-2, a Novel Member of the Inhibitor of Apoptosis Protein Family †

    doi: 10.1128/MCB.21.13.4292-4301.2001

    Figure Lengend Snippet: ILP-2 inhibits apoptosis induced by Bax and caspase 9 plus Apaf-1 but not by Fas. Human embryonic kidney 293 cells were transfected with a GFP plasmid (0.5 μg/well) in combination with either a Bax expression plasmid (0.25 μg/well) (A), Fas expression plasmid (2 μg/well) (B), or Apaf-1 (2 μg/well) and caspase 9 (0.5 μg/well) plasmids, as indicated (C). Cells were cotransfected with a plasmid encoding GFP as a marker of transfection, together with a control plasmid or plasmids expressing ILP-1, ILP-2, or dominant-negative caspase 9 (C-9 DN) (2 μg/well) as indicated. Cells were fixed and stained with DAPI, and transfected wells were scored for viability based on nuclear morphology as described in Materials and Methods. Data shown are representative of at least three independent experiments.

    Article Snippet: After blocking the membranes in 5% dry milk in Tris-buffered saline with 0.2% Tween, the membranes were incubated with either anti-GST antibody (Santa Cruz Laboratories, Santa Cruz, Calif.), anti-Apaf-1 antibody (a kind gift of Y. Lazebnik, Cold Spring Harbor, N.Y.), or anti-caspase 9 antibodies (Pharmingen, San Diego, Calif. or Upstate Biotechnology, Lake Placid, N.Y.) as indicated in the figure legends and were visualized by enhanced chemiluminescence (ECL; Amersham Pharmacia).

    Techniques: Transfection, Plasmid Preparation, Expressing, Marker, Dominant Negative Mutation, Staining

    ILP-2 inhibits caspase activation in a cell-free system. (A, B) Cytosolic extracts (150 μg) from 293 cells were preincubated with GST–ILP-1, GST–ILP-2, or GST control proteins (100 ng each) as indicated for 20 min, at which time ATP (1 mM final concentration) was added and the reaction mixture was incubated for a further 30 min. (C, D) Cytosolic extracts were incubated with ATP for 30 min, after which time the recombinant GST–ILP-1, GST–ILP-2, or GST control proteins were added and the reaction mixture was incubated for a further 20 min. Caspase activity was measured by cleavage of the caspase 3-specific fluorogenic substrate DEVD-AFC (A, C) or the caspase 9-specific substrate LEHD-AFC (B, D), as described in Materials and Methods.

    Journal: Molecular and Cellular Biology

    Article Title: Molecular Cloning of ILP-2, a Novel Member of the Inhibitor of Apoptosis Protein Family †

    doi: 10.1128/MCB.21.13.4292-4301.2001

    Figure Lengend Snippet: ILP-2 inhibits caspase activation in a cell-free system. (A, B) Cytosolic extracts (150 μg) from 293 cells were preincubated with GST–ILP-1, GST–ILP-2, or GST control proteins (100 ng each) as indicated for 20 min, at which time ATP (1 mM final concentration) was added and the reaction mixture was incubated for a further 30 min. (C, D) Cytosolic extracts were incubated with ATP for 30 min, after which time the recombinant GST–ILP-1, GST–ILP-2, or GST control proteins were added and the reaction mixture was incubated for a further 20 min. Caspase activity was measured by cleavage of the caspase 3-specific fluorogenic substrate DEVD-AFC (A, C) or the caspase 9-specific substrate LEHD-AFC (B, D), as described in Materials and Methods.

    Article Snippet: After blocking the membranes in 5% dry milk in Tris-buffered saline with 0.2% Tween, the membranes were incubated with either anti-GST antibody (Santa Cruz Laboratories, Santa Cruz, Calif.), anti-Apaf-1 antibody (a kind gift of Y. Lazebnik, Cold Spring Harbor, N.Y.), or anti-caspase 9 antibodies (Pharmingen, San Diego, Calif. or Upstate Biotechnology, Lake Placid, N.Y.) as indicated in the figure legends and were visualized by enhanced chemiluminescence (ECL; Amersham Pharmacia).

    Techniques: Activation Assay, Concentration Assay, Incubation, Recombinant, Activity Assay

     The time courses of proteolytic processing of pro-caspase 8 ( A ), Bid ( B ) and pro-caspase 9 ( C ) in MOLT-4 cells following intracytoplasmic delivery of CGTTA. Cells were loaded from an external concentration of 20 µM oligodeoxynucleotide and lysates were prepared at the indicated times after resuspension in fresh medium. Band intensities on western blots were determined by densitometry. The data were normalised to the values for cells treated with streptolysin O alone and each point represents the mean ± SD of three replicates.

    Journal: Nucleic Acids Research

    Article Title: Oligodeoxynucleotide 5mers containing a 5?-CpG induce apoptosis through a mitochondrial mechanism in T lymphocytic leukaemia cells

    doi:

    Figure Lengend Snippet: The time courses of proteolytic processing of pro-caspase 8 ( A ), Bid ( B ) and pro-caspase 9 ( C ) in MOLT-4 cells following intracytoplasmic delivery of CGTTA. Cells were loaded from an external concentration of 20 µM oligodeoxynucleotide and lysates were prepared at the indicated times after resuspension in fresh medium. Band intensities on western blots were determined by densitometry. The data were normalised to the values for cells treated with streptolysin O alone and each point represents the mean ± SD of three replicates.

    Article Snippet: Rabbit antibodies against human caspase 3, pro-caspase 8, pro-caspase 10, Bak and Bax were from Upstate Biotechnology (Waltham, MA; UK supplier TCS Biologicals Ltd, Buckingham, UK) and for human pro-caspase 9 and Bid from R & D Systems Europe Ltd.

    Techniques: Concentration Assay, Western Blot

    Bcr-Abl signaling does not induce phosphorylation of caspase 9. (A) Endogenous caspase 9 (cas-9) was immunoprecipitated from 293T cells, and in vitro kinase assays were performed with control GFP (lanes C)- or Bcr-Abl (lanes B)-expressing cell lysates supplemented with [γ- 32 P]ATP under conditions in which Bcr-Abl was autophosphorylated (bottom). Lysates were treated with recombinant active ERK (lanes E) as a control for caspase 9 phosphorylation. IgG, immunoglobulin G. (B) control GFP (lane C)- and Bcr-Abl (lane B)-expressing Rat-1 fibroblasts were radiolabeled with orthophosphate. Endogenous caspase 9 was captured with a GST fusion protein containing the CARD of Apaf-1. Caspase 9 bound to the Apaf-1 CARD was then subjected to SDS-PAGE and autoradiography or immunoblot analysis with an antibody directed against rat caspase 9. The values on the left are molecular size markers in kilodaltons.

    Journal: Molecular and Cellular Biology

    Article Title: Bcr-Abl-Mediated Protection from Apoptosis Downstream of Mitochondrial Cytochrome c Release

    doi: 10.1128/MCB.24.23.10289-10299.2004

    Figure Lengend Snippet: Bcr-Abl signaling does not induce phosphorylation of caspase 9. (A) Endogenous caspase 9 (cas-9) was immunoprecipitated from 293T cells, and in vitro kinase assays were performed with control GFP (lanes C)- or Bcr-Abl (lanes B)-expressing cell lysates supplemented with [γ- 32 P]ATP under conditions in which Bcr-Abl was autophosphorylated (bottom). Lysates were treated with recombinant active ERK (lanes E) as a control for caspase 9 phosphorylation. IgG, immunoglobulin G. (B) control GFP (lane C)- and Bcr-Abl (lane B)-expressing Rat-1 fibroblasts were radiolabeled with orthophosphate. Endogenous caspase 9 was captured with a GST fusion protein containing the CARD of Apaf-1. Caspase 9 bound to the Apaf-1 CARD was then subjected to SDS-PAGE and autoradiography or immunoblot analysis with an antibody directed against rat caspase 9. The values on the left are molecular size markers in kilodaltons.

    Article Snippet: Furthermore, several additional proteins have been identified which can inhibit apoptosis by binding to either Apaf-1 or caspase 9 (e.g., Hsp70 and Aven) to prevent proper functioning of the apoptosome ( - , , , ).

    Techniques: Immunoprecipitation, In Vitro, Expressing, Recombinant, SDS Page, Autoradiography

    Post-cytochrome c protection by Bcr-Abl in Xenopus egg extracts. (A) Crude egg extract was incubated with buffer alone, WT Bcr-Abl, or kinase-dead (K671R) Bcr-Abl, and caspase 3 activity was measured at various time points. Caspase 3 activity was measured spectrophotometrically via cleavage of the colorimetric caspase substrate DEVD-pNA. (B) Cytosolic egg extract was pretreated for 30 min with WT Bcr-Abl, K671R Bcr-Abl, or buffer alone and then incubated with 0.6 ng of cytochrome c per μl. Caspase 3 activity was measured by cleavage of DEVD-pNA over time. (C) Cytosolic egg extract was pretreated for 30 min with WT or K671R mutant Bcr-Abl and then incubated with 0.7 ng of cytochrome c per μl for various times. Caspase 9 activity was measured spectrophotometrically by cleavage of the colorimetric caspase 9 substrate LEHD-pNA. (D) In vitro-translated, 35 S-labeled procaspase 9 and soluble cytochrome c (0.6 ng/μl) were added to cytosolic egg extracts that were pretreated with WT or K671R mutant Bcr-Abl. Cleavage of procaspase 9 was assessed via autoradiography. In panels A to C, the graphs are representative of at least three independent experiments. OD405, absorbance reading at 405 nm.

    Journal: Molecular and Cellular Biology

    Article Title: Bcr-Abl-Mediated Protection from Apoptosis Downstream of Mitochondrial Cytochrome c Release

    doi: 10.1128/MCB.24.23.10289-10299.2004

    Figure Lengend Snippet: Post-cytochrome c protection by Bcr-Abl in Xenopus egg extracts. (A) Crude egg extract was incubated with buffer alone, WT Bcr-Abl, or kinase-dead (K671R) Bcr-Abl, and caspase 3 activity was measured at various time points. Caspase 3 activity was measured spectrophotometrically via cleavage of the colorimetric caspase substrate DEVD-pNA. (B) Cytosolic egg extract was pretreated for 30 min with WT Bcr-Abl, K671R Bcr-Abl, or buffer alone and then incubated with 0.6 ng of cytochrome c per μl. Caspase 3 activity was measured by cleavage of DEVD-pNA over time. (C) Cytosolic egg extract was pretreated for 30 min with WT or K671R mutant Bcr-Abl and then incubated with 0.7 ng of cytochrome c per μl for various times. Caspase 9 activity was measured spectrophotometrically by cleavage of the colorimetric caspase 9 substrate LEHD-pNA. (D) In vitro-translated, 35 S-labeled procaspase 9 and soluble cytochrome c (0.6 ng/μl) were added to cytosolic egg extracts that were pretreated with WT or K671R mutant Bcr-Abl. Cleavage of procaspase 9 was assessed via autoradiography. In panels A to C, the graphs are representative of at least three independent experiments. OD405, absorbance reading at 405 nm.

    Article Snippet: Furthermore, several additional proteins have been identified which can inhibit apoptosis by binding to either Apaf-1 or caspase 9 (e.g., Hsp70 and Aven) to prevent proper functioning of the apoptosome ( - , , , ).

    Techniques: Incubation, Activity Assay, Mutagenesis, In Vitro, Labeling, Autoradiography

    Bcr-Abl prevents processing of caspase 3 and caspase 9. (A) In vitro-translated, 35 S-radiolabeled, catalytically inactive procaspase 3 was added to control GFP- or Bcr-Abl-expressing Rat-1 lysates in the presence of soluble cytochrome c (1 ng/μl). The processing of caspase 3 was observed via autoradiography. (B) Cell lysates from control GFP- or Bcr-Abl-expressing Rat-1 fibroblasts were incubated with 1 mM dATP and various concentrations of soluble cytochrome c . The processing of endogenous caspase 9 was assayed by Western analysis. Note that the fragment derived from caspase 9-mediated caspase 9 cleavage (the middle of the three cleaved bands indicative that the apoptosome has been activated) is barely visible in the presence of Bcr-Abl. The small amount of (unsuppressed) residual caspase 9 activity in this experiment most likely led to caspase 3 activation, resulting in amplification of caspase 9 processing through caspase 3-mediated caspase 9 cleavage (the upper band of the triplet).

    Journal: Molecular and Cellular Biology

    Article Title: Bcr-Abl-Mediated Protection from Apoptosis Downstream of Mitochondrial Cytochrome c Release

    doi: 10.1128/MCB.24.23.10289-10299.2004

    Figure Lengend Snippet: Bcr-Abl prevents processing of caspase 3 and caspase 9. (A) In vitro-translated, 35 S-radiolabeled, catalytically inactive procaspase 3 was added to control GFP- or Bcr-Abl-expressing Rat-1 lysates in the presence of soluble cytochrome c (1 ng/μl). The processing of caspase 3 was observed via autoradiography. (B) Cell lysates from control GFP- or Bcr-Abl-expressing Rat-1 fibroblasts were incubated with 1 mM dATP and various concentrations of soluble cytochrome c . The processing of endogenous caspase 9 was assayed by Western analysis. Note that the fragment derived from caspase 9-mediated caspase 9 cleavage (the middle of the three cleaved bands indicative that the apoptosome has been activated) is barely visible in the presence of Bcr-Abl. The small amount of (unsuppressed) residual caspase 9 activity in this experiment most likely led to caspase 3 activation, resulting in amplification of caspase 9 processing through caspase 3-mediated caspase 9 cleavage (the upper band of the triplet).

    Article Snippet: Furthermore, several additional proteins have been identified which can inhibit apoptosis by binding to either Apaf-1 or caspase 9 (e.g., Hsp70 and Aven) to prevent proper functioning of the apoptosome ( - , , , ).

    Techniques: In Vitro, Expressing, Autoradiography, Incubation, Western Blot, Derivative Assay, Activity Assay, Activation Assay, Amplification

    Bcr-Abl post-cytochrome c protection in cell lysates occurs by a posttranslational mechanism. (A) Control GFP-expressing cell lysates were preincubated with Abl immunoprecipitates from control GFP- or Bcr-Abl-expressing Rat-1 cells, after which caspase activity was assessed following the addition of 1 ng of soluble cytochrome c per μl. Caspase 3 activity was measured by assessing the cleavage of the colorimetric DEVD-pNA substrate. The results shown are representative of three independent experiments. (B) Control GFP (lane C)- and Bcr-Abl (lane B)-expressing Rat-1 lysates were immunoblotted for the Apaf-1 and caspase 9 proteins. (C) Cytosolic Xenopus egg extracts were pretreated with Mos, WT Bcr-Abl (WT), or kinase-dead Bcr-Abl (K671R). ERK activation was assessed by Western immunoblot analysis with an antibody directed against phospho-ERK. OD405, absorbance reading at 405 nm.

    Journal: Molecular and Cellular Biology

    Article Title: Bcr-Abl-Mediated Protection from Apoptosis Downstream of Mitochondrial Cytochrome c Release

    doi: 10.1128/MCB.24.23.10289-10299.2004

    Figure Lengend Snippet: Bcr-Abl post-cytochrome c protection in cell lysates occurs by a posttranslational mechanism. (A) Control GFP-expressing cell lysates were preincubated with Abl immunoprecipitates from control GFP- or Bcr-Abl-expressing Rat-1 cells, after which caspase activity was assessed following the addition of 1 ng of soluble cytochrome c per μl. Caspase 3 activity was measured by assessing the cleavage of the colorimetric DEVD-pNA substrate. The results shown are representative of three independent experiments. (B) Control GFP (lane C)- and Bcr-Abl (lane B)-expressing Rat-1 lysates were immunoblotted for the Apaf-1 and caspase 9 proteins. (C) Cytosolic Xenopus egg extracts were pretreated with Mos, WT Bcr-Abl (WT), or kinase-dead Bcr-Abl (K671R). ERK activation was assessed by Western immunoblot analysis with an antibody directed against phospho-ERK. OD405, absorbance reading at 405 nm.

    Article Snippet: Furthermore, several additional proteins have been identified which can inhibit apoptosis by binding to either Apaf-1 or caspase 9 (e.g., Hsp70 and Aven) to prevent proper functioning of the apoptosome ( - , , , ).

    Techniques: Expressing, Activity Assay, Activation Assay, Western Blot

    Bcr-Abl inhibits caspase 9 recruitment to the apoptosome. GST fusion proteins containing the prodomain of caspase 9 (A), the CARD of Apaf-1 (B), or Apaf-1 (1-543) (G) were incubated with control GFP (lanes C)- or Bcr-Abl (lanes B)-expressing lysates in the presence of 1 mM dATP. GST proteins were then rebound to glutathione beads (GSH), and the binding ability of Apaf-1 (A) or caspase 9 (B and G) was examined via immunoblot analysis. (C) GST-Apaf-1 (1-543), GST, or GST-Crk protein was incubated with [γ- 32 P]ATP in either control GFP (lane C)- or Bcr-Abl (lane B)-expressing cell lysates, and radiolabeling was assessed via autoradiography. (D) Apaf-1 was immunoprecipitated from either control GFP (lane C)- or Bcr-Abl (lane B)-expressing lysates, and precipitates were immunoblotted (IB) for Apaf-1 or phosphotyrosine (P-Tyr). Anti-Abl immunoprecipitates (IP) were also immunoblotted for P-Tyr as a positive control for the phosphotyrosine Western analysis. (E and F) Control GFP (lane C)- or Bcr-Abl (lane B)-expressing lysates were incubated with cytochrome c -Sepharose and 1 mM dATP. The binding ability of Apaf-1 (E) and caspase 9 (F) was then examined by immunoblot analysis.

    Journal: Molecular and Cellular Biology

    Article Title: Bcr-Abl-Mediated Protection from Apoptosis Downstream of Mitochondrial Cytochrome c Release

    doi: 10.1128/MCB.24.23.10289-10299.2004

    Figure Lengend Snippet: Bcr-Abl inhibits caspase 9 recruitment to the apoptosome. GST fusion proteins containing the prodomain of caspase 9 (A), the CARD of Apaf-1 (B), or Apaf-1 (1-543) (G) were incubated with control GFP (lanes C)- or Bcr-Abl (lanes B)-expressing lysates in the presence of 1 mM dATP. GST proteins were then rebound to glutathione beads (GSH), and the binding ability of Apaf-1 (A) or caspase 9 (B and G) was examined via immunoblot analysis. (C) GST-Apaf-1 (1-543), GST, or GST-Crk protein was incubated with [γ- 32 P]ATP in either control GFP (lane C)- or Bcr-Abl (lane B)-expressing cell lysates, and radiolabeling was assessed via autoradiography. (D) Apaf-1 was immunoprecipitated from either control GFP (lane C)- or Bcr-Abl (lane B)-expressing lysates, and precipitates were immunoblotted (IB) for Apaf-1 or phosphotyrosine (P-Tyr). Anti-Abl immunoprecipitates (IP) were also immunoblotted for P-Tyr as a positive control for the phosphotyrosine Western analysis. (E and F) Control GFP (lane C)- or Bcr-Abl (lane B)-expressing lysates were incubated with cytochrome c -Sepharose and 1 mM dATP. The binding ability of Apaf-1 (E) and caspase 9 (F) was then examined by immunoblot analysis.

    Article Snippet: Furthermore, several additional proteins have been identified which can inhibit apoptosis by binding to either Apaf-1 or caspase 9 (e.g., Hsp70 and Aven) to prevent proper functioning of the apoptosome ( - , , , ).

    Techniques: Incubation, Expressing, Binding Assay, Radioactivity, Autoradiography, Immunoprecipitation, Positive Control, Western Blot

    Identification of the Z-VAD– binding activity as caspase-2 and -9. (A) Proteins reacting with biotinylated VAD.fmk in the intermembrane space of liver mitochondria. The supernatant of control mitochondria (lane 1) or of Atr-treated mitochondria (lanes 2 and 3), the flow-through of the MiniS column (see Fig. 2 and Fig. 3 A; lanes 4 and 5), purified AIF (lane 6), or the Z-VAD.afc–cleaving activity eluting at 280 mM from the MiniQ column (see Fig. 2 and Fig. 4 A; lanes 7 and 8) were allowed to react with biotinylated VAD.fmk, either without pretreatment (lanes 1, 2, 4, 6, and 7) or after preincubation with Z-VAD.fmk (lanes 3, 5, and 8). Note that Z-VAD.fmk has been added to mitochondria before Atr (line 3). In addition, the proteins reacting with biotinylated VAD.fmk retained on an avidin column were purified (lane 9). These proteins, which contained approximately similar levels of Z-VAD.afc–cleaving activity (10 U) or ∼100 ng purified protein (lane 6) were separated by SDS-PAGE, blotted onto nitrocellulose, and subjected to the detection of biotinylated VAD.fmk using an avidin-based detection system. (B and C) The same blot as in A was subjected to immunodetection with antibodies specific for caspase-2 (B) or -9 (C). (D and E) Mitochondria from different organs were purified and cultured for 30 min in the presence or absence of 5 mM Atr, followed by immunoblot detection of caspase-2 (D) or -9 (E). Results are representative of two to four independent experiments. (F and G) Specificity control of caspase-2– and caspase-9–specific antisera. Recombinant caspase-2 (lane 1), -3 (lane 2), or -9 (lane 3) was immunoblotted (100 ng/lane), followed by immunodetection with the caspase-2 (F) or caspase-9 (G)–specific antibody. Similarly, caspase-2– and caspase-9–specific antibodies fail to recognize caspase-6 and -7 (not shown).

    Journal: The Journal of Experimental Medicine

    Article Title: Mitochondrial Release of Caspase-2 and -9 during the Apoptotic Process

    doi:

    Figure Lengend Snippet: Identification of the Z-VAD– binding activity as caspase-2 and -9. (A) Proteins reacting with biotinylated VAD.fmk in the intermembrane space of liver mitochondria. The supernatant of control mitochondria (lane 1) or of Atr-treated mitochondria (lanes 2 and 3), the flow-through of the MiniS column (see Fig. 2 and Fig. 3 A; lanes 4 and 5), purified AIF (lane 6), or the Z-VAD.afc–cleaving activity eluting at 280 mM from the MiniQ column (see Fig. 2 and Fig. 4 A; lanes 7 and 8) were allowed to react with biotinylated VAD.fmk, either without pretreatment (lanes 1, 2, 4, 6, and 7) or after preincubation with Z-VAD.fmk (lanes 3, 5, and 8). Note that Z-VAD.fmk has been added to mitochondria before Atr (line 3). In addition, the proteins reacting with biotinylated VAD.fmk retained on an avidin column were purified (lane 9). These proteins, which contained approximately similar levels of Z-VAD.afc–cleaving activity (10 U) or ∼100 ng purified protein (lane 6) were separated by SDS-PAGE, blotted onto nitrocellulose, and subjected to the detection of biotinylated VAD.fmk using an avidin-based detection system. (B and C) The same blot as in A was subjected to immunodetection with antibodies specific for caspase-2 (B) or -9 (C). (D and E) Mitochondria from different organs were purified and cultured for 30 min in the presence or absence of 5 mM Atr, followed by immunoblot detection of caspase-2 (D) or -9 (E). Results are representative of two to four independent experiments. (F and G) Specificity control of caspase-2– and caspase-9–specific antisera. Recombinant caspase-2 (lane 1), -3 (lane 2), or -9 (lane 3) was immunoblotted (100 ng/lane), followed by immunodetection with the caspase-2 (F) or caspase-9 (G)–specific antibody. Similarly, caspase-2– and caspase-9–specific antibodies fail to recognize caspase-6 and -7 (not shown).

    Article Snippet: In addition, we found that crude supernatants of Atr-treated mitochondria, purified Z-VADase, and the Z-VAD.biotin–binding activity reacted with an antiserum specific for the large 18-kD subunit of caspase-9 (which recognize the 48-kD procaspase, the 33-kD intermediate, and the 18-kD large subunit of the mature caspase-9) (Fig. C).

    Techniques: Binding Assay, Activity Assay, Flow Cytometry, Purification, Avidin-Biotin Assay, SDS Page, Immunodetection, Cell Culture, Recombinant

    Apoptosis induction by microinjection of recombinant caspases. Rat-1 fibroblasts were left untreated or cultured with etoposide (1 μM, 6 h), in the absence or presence of Z-VAD.fmk (100 μM), to obtain a positive or negative control of apoptotic morphology, respectively. Alternatively, cells pretreated with Z-VAD.fmk (100 μM, 60 min) or left untreated were microinjected with buffer only or with recombinant caspase-2 or -9 (3 U/μl). Microinjected viable cells (100–200 per session, two to five independent sessions of injection) could be identified because the injectate contained FITC-dextran (0.25% [wt/vol], green fluorescence; not shown). Microphotographs representing the dominant ( > 70%) phenotype of microinjected cells stained either with Hoechst 33342 (blue fluorescence, A) or with Annexin V (red fluorescence, B) are shown after 90 min of culture. Z-VAD. fmk pretreatment prevented phosphatidylserine exposure induced by etoposide, caspase-2, or caspase-9, as measured with Annexin V (not shown).

    Journal: The Journal of Experimental Medicine

    Article Title: Mitochondrial Release of Caspase-2 and -9 during the Apoptotic Process

    doi:

    Figure Lengend Snippet: Apoptosis induction by microinjection of recombinant caspases. Rat-1 fibroblasts were left untreated or cultured with etoposide (1 μM, 6 h), in the absence or presence of Z-VAD.fmk (100 μM), to obtain a positive or negative control of apoptotic morphology, respectively. Alternatively, cells pretreated with Z-VAD.fmk (100 μM, 60 min) or left untreated were microinjected with buffer only or with recombinant caspase-2 or -9 (3 U/μl). Microinjected viable cells (100–200 per session, two to five independent sessions of injection) could be identified because the injectate contained FITC-dextran (0.25% [wt/vol], green fluorescence; not shown). Microphotographs representing the dominant ( > 70%) phenotype of microinjected cells stained either with Hoechst 33342 (blue fluorescence, A) or with Annexin V (red fluorescence, B) are shown after 90 min of culture. Z-VAD. fmk pretreatment prevented phosphatidylserine exposure induced by etoposide, caspase-2, or caspase-9, as measured with Annexin V (not shown).

    Article Snippet: In addition, we found that crude supernatants of Atr-treated mitochondria, purified Z-VADase, and the Z-VAD.biotin–binding activity reacted with an antiserum specific for the large 18-kD subunit of caspase-9 (which recognize the 48-kD procaspase, the 33-kD intermediate, and the 18-kD large subunit of the mature caspase-9) (Fig. C).

    Techniques: Recombinant, Cell Culture, Negative Control, Injection, Fluorescence, Staining

    Loss of subcellular compartmentalization of cytochrome c , caspase-2, and caspase-9. Rat-1 cells were left untreated or were cultured in the presence of staurosporine (4 h, 1 μM), fixed, and stained to determine the subcellular distribution of the indicated molecules by indirect immunofluorescence analysis.

    Journal: The Journal of Experimental Medicine

    Article Title: Mitochondrial Release of Caspase-2 and -9 during the Apoptotic Process

    doi:

    Figure Lengend Snippet: Loss of subcellular compartmentalization of cytochrome c , caspase-2, and caspase-9. Rat-1 cells were left untreated or were cultured in the presence of staurosporine (4 h, 1 μM), fixed, and stained to determine the subcellular distribution of the indicated molecules by indirect immunofluorescence analysis.

    Article Snippet: In addition, we found that crude supernatants of Atr-treated mitochondria, purified Z-VADase, and the Z-VAD.biotin–binding activity reacted with an antiserum specific for the large 18-kD subunit of caspase-9 (which recognize the 48-kD procaspase, the 33-kD intermediate, and the 18-kD large subunit of the mature caspase-9) (Fig. C).

    Techniques: Cell Culture, Staining, Immunofluorescence

    Submitochondrial localization of caspase-2 and -9. Purified mitochondria were either lysed by osmotic shock (total preparation, lane 1) or were subjected to fractionation into matrix (lanes 2 and 6), inner membrane (lanes 3 and 7), intermembrane (lanes 4 and 8), or outer membrane (lanes 5 and 9) proteins, in the absence (lanes 2–5) or presence (lanes 6–9) of 100 μM Z-VAD.fmk throughout each single step of the fractionation procedure. Thereafter, equivalent amounts of protein (15 μg/lane) were subjected to immunoblot detection of caspase-2 (A) and -9 (B). A representative immunoelectron micrograph of mitochondria labeled with a specific caspase-9 antibody is also shown (C).

    Journal: The Journal of Experimental Medicine

    Article Title: Mitochondrial Release of Caspase-2 and -9 during the Apoptotic Process

    doi:

    Figure Lengend Snippet: Submitochondrial localization of caspase-2 and -9. Purified mitochondria were either lysed by osmotic shock (total preparation, lane 1) or were subjected to fractionation into matrix (lanes 2 and 6), inner membrane (lanes 3 and 7), intermembrane (lanes 4 and 8), or outer membrane (lanes 5 and 9) proteins, in the absence (lanes 2–5) or presence (lanes 6–9) of 100 μM Z-VAD.fmk throughout each single step of the fractionation procedure. Thereafter, equivalent amounts of protein (15 μg/lane) were subjected to immunoblot detection of caspase-2 (A) and -9 (B). A representative immunoelectron micrograph of mitochondria labeled with a specific caspase-9 antibody is also shown (C).

    Article Snippet: In addition, we found that crude supernatants of Atr-treated mitochondria, purified Z-VADase, and the Z-VAD.biotin–binding activity reacted with an antiserum specific for the large 18-kD subunit of caspase-9 (which recognize the 48-kD procaspase, the 33-kD intermediate, and the 18-kD large subunit of the mature caspase-9) (Fig. C).

    Techniques: Purification, Fractionation, Labeling

    Redistribution of caspase-2 and -9 from the mitochondrion. T cell hybridoma cells transfected with a Neo control vector or with Bcl-2 were cultured in the absence or presence of 1 μM DEX, followed by subcellular fractionation, as described in Materials and Methods. Equivalent amounts of proteins were subjected to immunoblot analysis in order to determine the subcellular localization and activation of cytochrome c (A), caspase-2 (B), or caspase-9 (C). Results are representative of three independent determinations.

    Journal: The Journal of Experimental Medicine

    Article Title: Mitochondrial Release of Caspase-2 and -9 during the Apoptotic Process

    doi:

    Figure Lengend Snippet: Redistribution of caspase-2 and -9 from the mitochondrion. T cell hybridoma cells transfected with a Neo control vector or with Bcl-2 were cultured in the absence or presence of 1 μM DEX, followed by subcellular fractionation, as described in Materials and Methods. Equivalent amounts of proteins were subjected to immunoblot analysis in order to determine the subcellular localization and activation of cytochrome c (A), caspase-2 (B), or caspase-9 (C). Results are representative of three independent determinations.

    Article Snippet: In addition, we found that crude supernatants of Atr-treated mitochondria, purified Z-VADase, and the Z-VAD.biotin–binding activity reacted with an antiserum specific for the large 18-kD subunit of caspase-9 (which recognize the 48-kD procaspase, the 33-kD intermediate, and the 18-kD large subunit of the mature caspase-9) (Fig. C).

    Techniques: Transfection, Plasmid Preparation, Cell Culture, Fractionation, Activation Assay

    A novel pathway of Tom20-Bax-caspase-GSDME upon iron stimulation. In melanoma cells, iron-elevated ROS causes the oxidation and oligomerization of Tom20. Oxidized Tom20 induces Bax translocation to mitochondria, which facilitates cytochrome c release to cytosol. Once released, cytochrome c activates caspase-9, which then activates caspase-3. This caspase-3 activation further cleaves GSDME, and eventually triggers cell swelling and LDH release

    Journal: Cell Research

    Article Title: Tom20 senses iron-activated ROS signaling to promote melanoma cell pyroptosis

    doi: 10.1038/s41422-018-0090-y

    Figure Lengend Snippet: A novel pathway of Tom20-Bax-caspase-GSDME upon iron stimulation. In melanoma cells, iron-elevated ROS causes the oxidation and oligomerization of Tom20. Oxidized Tom20 induces Bax translocation to mitochondria, which facilitates cytochrome c release to cytosol. Once released, cytochrome c activates caspase-9, which then activates caspase-3. This caspase-3 activation further cleaves GSDME, and eventually triggers cell swelling and LDH release

    Article Snippet: Recombinant active Caspase-1 (Cat# ALX-201-056-U025), Caspase-7 (Cat# ALX-201-061-U025), Caspase-8 (Cat# ALX-201-062-U025) and Caspase-9 (Cat# ALX-201-047-U025) were from Enzo Life Sciences.

    Techniques: Translocation Assay, Activation Assay