human recombinant caspase-6 Search Results


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  • 99
    Millipore recombinant human caspase 6
    Recombinant Human Caspase 6, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant human caspase 6/product/Millipore
    Average 99 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    recombinant human caspase 6 - by Bioz Stars, 2020-04
    99/100 stars
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    95
    Enzo Biochem human recombinant caspase 6
    Degradation of macrophage cytoskeletal-associated proteins upon contact with Eh is mediated by <t>caspase-6.</t> ( a ) THP-1 macrophages were pre-treated with increasing amounts of the pan-caspase inhibitor Z-VAD-fmk or DMSO as control, for 1 h prior to incubation with Eh (20:1 ratio) for 5 min. Cleavage of the indicated cytoskeletal proteins was assessed by Western blot. ( b ) THP-1 macrophages were pre-treated with 50μM Z-VAD-fmk, 50μM of the caspase-6 inhibitor Z-VEID-fmk or DMSO as a control, followed by incubation with Eh (20:1) for 5 min. ( c ) Immunoblot of caspase-6 and its lamin A/C in macrophages incubated with Eh (20:1) for the increasing incubation times. Dose-dependent inhibition of cleavage of the indicated cytoskeletal proteins ( d ) or caspase-6 and its substrate ( e ) by Z-VEID. To inhibit calpain activation in macrophages, cells were pretreated with E-64 prior to incubation with Eh (10:1) and cell lysis ( f ). Lysates were loaded onto SDS-PAGE and immunoblotted with the indicated antibodies. ( g ) THP-1 cells were transfected with 25 and 50nM of caspase-6 siRNA, or scramble siRNA. After 48 h, transfected cells were stimulated for 2 min with Eh , followed by Western blot with the indicated antibodies. ( h ) Direct cleavage of paxillin was assessed by incubated immunoprecipitated paxillin with recombinant caspase-6 (C-6), with or without Z-VEID. Immunoprecipitated lamin was used as a positive control.
    Human Recombinant Caspase 6, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 95/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human recombinant caspase 6/product/Enzo Biochem
    Average 95 stars, based on 14 article reviews
    Price from $9.99 to $1999.99
    human recombinant caspase 6 - by Bioz Stars, 2020-04
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    84
    Genentech cleavage assay active human recombinant caspase 6
    Western blot detection of recombinant GST-lamin A processing by purified caspases. (A) The indicated concentration of <t>caspase-6</t> was incubated with GST-lamin A for two hours. (B) 300 Units of caspases 1–9 were incubated with GST-lamin A for two hours. Intact and cleaved lamin A were detected via western blotting using anti-GST antibody.
    Cleavage Assay Active Human Recombinant Caspase 6, supplied by Genentech, used in various techniques. Bioz Stars score: 84/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cleavage assay active human recombinant caspase 6/product/Genentech
    Average 84 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    cleavage assay active human recombinant caspase 6 - by Bioz Stars, 2020-04
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    99
    BioVision intratracheal active recombinant human caspase 6
    Characterization of caspase cleavage site-specific antibody to N-GFAP ( A ) Caspase-cleaved GFAP was analyzed by immunoblotting with the indicated antibodies. The SMI-21 antibody recognized both intact GFAP and N-GFAP (lane 1), whereas the D225 antibody recognized N-GFAP but not intact protein (lane 2). ( B ) The D225 antibody also recognized purified recombinant N-GFAP (lane 1) but not C-GFAP ( B , lane 2), confirming the specificity of this antibody. ( C ) The presence of C-GFAP was demonstrated by immunoblotting with GA-5 antibody (lane 2). ( D ) Purified recombinant human (lane 1) and mouse GFAP (lane 3) digested with active <t>caspase</t> 6 (lanes 2 and 4) were analyzed by immunoblotting. Notice that the D225 antibody recognized caspase-generated N-terminal fragments from both human (lane 2) and mouse GFAP (lane 4).
    Intratracheal Active Recombinant Human Caspase 6, supplied by BioVision, used in various techniques. Bioz Stars score: 99/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/intratracheal active recombinant human caspase 6/product/BioVision
    Average 99 stars, based on 13 article reviews
    Price from $9.99 to $1999.99
    intratracheal active recombinant human caspase 6 - by Bioz Stars, 2020-04
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    91
    Becton Dickinson recombinant human caspase 6
    Characterization of caspase cleavage site-specific antibody to N-GFAP ( A ) Caspase-cleaved GFAP was analyzed by immunoblotting with the indicated antibodies. The SMI-21 antibody recognized both intact GFAP and N-GFAP (lane 1), whereas the D225 antibody recognized N-GFAP but not intact protein (lane 2). ( B ) The D225 antibody also recognized purified recombinant N-GFAP (lane 1) but not C-GFAP ( B , lane 2), confirming the specificity of this antibody. ( C ) The presence of C-GFAP was demonstrated by immunoblotting with GA-5 antibody (lane 2). ( D ) Purified recombinant human (lane 1) and mouse GFAP (lane 3) digested with active <t>caspase</t> 6 (lanes 2 and 4) were analyzed by immunoblotting. Notice that the D225 antibody recognized caspase-generated N-terminal fragments from both human (lane 2) and mouse GFAP (lane 4).
    Recombinant Human Caspase 6, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant human caspase 6/product/Becton Dickinson
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    recombinant human caspase 6 - by Bioz Stars, 2020-04
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    94
    Abcam recombinant full length active human caspase 6 protein
    Characterization of caspase cleavage site-specific antibody to N-GFAP ( A ) Caspase-cleaved GFAP was analyzed by immunoblotting with the indicated antibodies. The SMI-21 antibody recognized both intact GFAP and N-GFAP (lane 1), whereas the D225 antibody recognized N-GFAP but not intact protein (lane 2). ( B ) The D225 antibody also recognized purified recombinant N-GFAP (lane 1) but not C-GFAP ( B , lane 2), confirming the specificity of this antibody. ( C ) The presence of C-GFAP was demonstrated by immunoblotting with GA-5 antibody (lane 2). ( D ) Purified recombinant human (lane 1) and mouse GFAP (lane 3) digested with active <t>caspase</t> 6 (lanes 2 and 4) were analyzed by immunoblotting. Notice that the D225 antibody recognized caspase-generated N-terminal fragments from both human (lane 2) and mouse GFAP (lane 4).
    Recombinant Full Length Active Human Caspase 6 Protein, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant full length active human caspase 6 protein/product/Abcam
    Average 94 stars, based on 2 article reviews
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    99
    Cell Signaling Technology Inc caspase 6
    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 <t>caspase-6</t> (right panel) and the activities of the respective caspases were measured after 1 h incubation.
    Caspase 6, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 193 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/caspase 6/product/Cell Signaling Technology Inc
    Average 99 stars, based on 193 article reviews
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    caspase 6 - by Bioz Stars, 2020-04
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    84
    Biomol GmbH caspase 6
    Astrocytes are resistant to <t>caspase-6,</t> -7, and -8 but undergo apoptosis with caspase-3. Astrocytes were injected with 20 or 100 pg/cell. Apoptosis was determined by TUNEL. Data represents the mean and SD of two experiments from three astrocyte preparations.
    Caspase 6, supplied by Biomol GmbH, used in various techniques. Bioz Stars score: 84/100, based on 72 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/caspase 6/product/Biomol GmbH
    Average 84 stars, based on 72 article reviews
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    caspase 6 - by Bioz Stars, 2020-04
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    99
    Cell Signaling Technology Inc procaspase antibody sampler kit
    Astrocytes are resistant to <t>caspase-6,</t> -7, and -8 but undergo apoptosis with caspase-3. Astrocytes were injected with 20 or 100 pg/cell. Apoptosis was determined by TUNEL. Data represents the mean and SD of two experiments from three astrocyte preparations.
    Procaspase Antibody Sampler Kit, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/procaspase antibody sampler kit/product/Cell Signaling Technology Inc
    Average 99 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
    procaspase antibody sampler kit - by Bioz Stars, 2020-04
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    Image Search Results


    Degradation of macrophage cytoskeletal-associated proteins upon contact with Eh is mediated by caspase-6. ( a ) THP-1 macrophages were pre-treated with increasing amounts of the pan-caspase inhibitor Z-VAD-fmk or DMSO as control, for 1 h prior to incubation with Eh (20:1 ratio) for 5 min. Cleavage of the indicated cytoskeletal proteins was assessed by Western blot. ( b ) THP-1 macrophages were pre-treated with 50μM Z-VAD-fmk, 50μM of the caspase-6 inhibitor Z-VEID-fmk or DMSO as a control, followed by incubation with Eh (20:1) for 5 min. ( c ) Immunoblot of caspase-6 and its lamin A/C in macrophages incubated with Eh (20:1) for the increasing incubation times. Dose-dependent inhibition of cleavage of the indicated cytoskeletal proteins ( d ) or caspase-6 and its substrate ( e ) by Z-VEID. To inhibit calpain activation in macrophages, cells were pretreated with E-64 prior to incubation with Eh (10:1) and cell lysis ( f ). Lysates were loaded onto SDS-PAGE and immunoblotted with the indicated antibodies. ( g ) THP-1 cells were transfected with 25 and 50nM of caspase-6 siRNA, or scramble siRNA. After 48 h, transfected cells were stimulated for 2 min with Eh , followed by Western blot with the indicated antibodies. ( h ) Direct cleavage of paxillin was assessed by incubated immunoprecipitated paxillin with recombinant caspase-6 (C-6), with or without Z-VEID. Immunoprecipitated lamin was used as a positive control.

    Journal: PLoS Pathogens

    Article Title: The macrophage cytoskeleton acts as a contact sensor upon interaction with Entamoeba histolytica to trigger IL-1β secretion

    doi: 10.1371/journal.ppat.1006592

    Figure Lengend Snippet: Degradation of macrophage cytoskeletal-associated proteins upon contact with Eh is mediated by caspase-6. ( a ) THP-1 macrophages were pre-treated with increasing amounts of the pan-caspase inhibitor Z-VAD-fmk or DMSO as control, for 1 h prior to incubation with Eh (20:1 ratio) for 5 min. Cleavage of the indicated cytoskeletal proteins was assessed by Western blot. ( b ) THP-1 macrophages were pre-treated with 50μM Z-VAD-fmk, 50μM of the caspase-6 inhibitor Z-VEID-fmk or DMSO as a control, followed by incubation with Eh (20:1) for 5 min. ( c ) Immunoblot of caspase-6 and its lamin A/C in macrophages incubated with Eh (20:1) for the increasing incubation times. Dose-dependent inhibition of cleavage of the indicated cytoskeletal proteins ( d ) or caspase-6 and its substrate ( e ) by Z-VEID. To inhibit calpain activation in macrophages, cells were pretreated with E-64 prior to incubation with Eh (10:1) and cell lysis ( f ). Lysates were loaded onto SDS-PAGE and immunoblotted with the indicated antibodies. ( g ) THP-1 cells were transfected with 25 and 50nM of caspase-6 siRNA, or scramble siRNA. After 48 h, transfected cells were stimulated for 2 min with Eh , followed by Western blot with the indicated antibodies. ( h ) Direct cleavage of paxillin was assessed by incubated immunoprecipitated paxillin with recombinant caspase-6 (C-6), with or without Z-VEID. Immunoprecipitated lamin was used as a positive control.

    Article Snippet: Recombinant human caspase-6 and the Z-VVR-AMC substrate were purchased from Enzo Life Sciences.

    Techniques: Incubation, Western Blot, Inhibition, Activation Assay, Lysis, SDS Page, Transfection, Immunoprecipitation, Recombinant, Positive Control

    Inhibition of caspase-6 or Eh CP-A1 and Eh CP-A4 reduces IL-1β secretion by macrophages in response to Eh . Caspase-6 was inhibited by pre-treatment of macrophages with 50mM of Z-VEID, or DMSO as control, prior to incubation with Eh (10:1 ratio) for 60 min. Eh CP-A1 and Eh CP-A4 were inhibited by pre-treatment of Eh with 20 μM of WRR483, or WRR605, or both, for 30 minutes prior to incubation with macrophages for 60 min. Both IL-1β secretion ( a ) and LDH release ( b ) were measured in media by ELISA. Results are representative of three independent experiments. Statistical significance was assessed by Student’s t -test (**p

    Journal: PLoS Pathogens

    Article Title: The macrophage cytoskeleton acts as a contact sensor upon interaction with Entamoeba histolytica to trigger IL-1β secretion

    doi: 10.1371/journal.ppat.1006592

    Figure Lengend Snippet: Inhibition of caspase-6 or Eh CP-A1 and Eh CP-A4 reduces IL-1β secretion by macrophages in response to Eh . Caspase-6 was inhibited by pre-treatment of macrophages with 50mM of Z-VEID, or DMSO as control, prior to incubation with Eh (10:1 ratio) for 60 min. Eh CP-A1 and Eh CP-A4 were inhibited by pre-treatment of Eh with 20 μM of WRR483, or WRR605, or both, for 30 minutes prior to incubation with macrophages for 60 min. Both IL-1β secretion ( a ) and LDH release ( b ) were measured in media by ELISA. Results are representative of three independent experiments. Statistical significance was assessed by Student’s t -test (**p

    Article Snippet: Recombinant human caspase-6 and the Z-VVR-AMC substrate were purchased from Enzo Life Sciences.

    Techniques: Inhibition, Incubation, Enzyme-linked Immunosorbent Assay

    Schematic representation of the Eh -macrophage interacting proteins at the intercellular junction. Contact between Eh and macrophages are initiated by high affinity binding with the Gal-lectin. Eh CP-A5, which is expressed at the surface of Eh , has been shown to bind to the α 5 β 1 integrin at the surface of macrophages through the RGD motif. This in turn activates the NLRP3 inflammasome and triggers IL-1β secretion by macrophages. In parallel, Eh CP-A1 and Eh CP-A4, which are localized in intracellular vesicles, are released at the intercellular junction. Through an unidentified mechanism, these cysteine proteinases trigger caspase-6 activation and subsequent cleavage of cytoskeletal-associated proteins paxillin, Pyk2 and talin. This arm of the Eh -macrophage interaction contributes to enhance IL-1β release.

    Journal: PLoS Pathogens

    Article Title: The macrophage cytoskeleton acts as a contact sensor upon interaction with Entamoeba histolytica to trigger IL-1β secretion

    doi: 10.1371/journal.ppat.1006592

    Figure Lengend Snippet: Schematic representation of the Eh -macrophage interacting proteins at the intercellular junction. Contact between Eh and macrophages are initiated by high affinity binding with the Gal-lectin. Eh CP-A5, which is expressed at the surface of Eh , has been shown to bind to the α 5 β 1 integrin at the surface of macrophages through the RGD motif. This in turn activates the NLRP3 inflammasome and triggers IL-1β secretion by macrophages. In parallel, Eh CP-A1 and Eh CP-A4, which are localized in intracellular vesicles, are released at the intercellular junction. Through an unidentified mechanism, these cysteine proteinases trigger caspase-6 activation and subsequent cleavage of cytoskeletal-associated proteins paxillin, Pyk2 and talin. This arm of the Eh -macrophage interaction contributes to enhance IL-1β release.

    Article Snippet: Recombinant human caspase-6 and the Z-VVR-AMC substrate were purchased from Enzo Life Sciences.

    Techniques: Binding Assay, Activation Assay

    Eh -triggered caspase-6 activation is dependent on Eh CP-A1 and Eh CP-A4 and is localized at sites of contact. ( a ) Caspase-6 activation was measured by flow cytometry with the FITC-labeled caspase-6-FLICA probe in THP-1 macrophages incubated with 10:1 Eh , E64-treated Eh , CP-A5-deficient Eh , WRR483 pre-treated Eh , WRR605 pre-treated Eh , Eh pre-treated with both WRR483 and WRR605, left untreated, or with STS as a positive control. Eh were excluded from analysis based on forward and side scatter. Gating of percent positive cells was established according to the STS-positive population and percent positive cells for each treatment are indicated in the accompanying graph. ( b ) Caspase-6 activation for each treatment was also visualized by confocal microscopy using caspase-6-FLICA (green), DAPI (blue). Eh were pre-treated with Cell Tracker Orange (red) prior to incubation with THP-1 macrophages. STS was used as a positive control. Arrowheads show areas of polarization of caspase-6 towards the Eh -macrophage interface. The dotted white line indicates the position of Eh and the point of contact with the macrophage (MΦ). Results are representative of three independent experiments.

    Journal: PLoS Pathogens

    Article Title: The macrophage cytoskeleton acts as a contact sensor upon interaction with Entamoeba histolytica to trigger IL-1β secretion

    doi: 10.1371/journal.ppat.1006592

    Figure Lengend Snippet: Eh -triggered caspase-6 activation is dependent on Eh CP-A1 and Eh CP-A4 and is localized at sites of contact. ( a ) Caspase-6 activation was measured by flow cytometry with the FITC-labeled caspase-6-FLICA probe in THP-1 macrophages incubated with 10:1 Eh , E64-treated Eh , CP-A5-deficient Eh , WRR483 pre-treated Eh , WRR605 pre-treated Eh , Eh pre-treated with both WRR483 and WRR605, left untreated, or with STS as a positive control. Eh were excluded from analysis based on forward and side scatter. Gating of percent positive cells was established according to the STS-positive population and percent positive cells for each treatment are indicated in the accompanying graph. ( b ) Caspase-6 activation for each treatment was also visualized by confocal microscopy using caspase-6-FLICA (green), DAPI (blue). Eh were pre-treated with Cell Tracker Orange (red) prior to incubation with THP-1 macrophages. STS was used as a positive control. Arrowheads show areas of polarization of caspase-6 towards the Eh -macrophage interface. The dotted white line indicates the position of Eh and the point of contact with the macrophage (MΦ). Results are representative of three independent experiments.

    Article Snippet: Recombinant human caspase-6 and the Z-VVR-AMC substrate were purchased from Enzo Life Sciences.

    Techniques: Activation Assay, Flow Cytometry, Cytometry, Labeling, Incubation, Positive Control, Confocal Microscopy

    Western blot detection of recombinant GST-lamin A processing by purified caspases. (A) The indicated concentration of caspase-6 was incubated with GST-lamin A for two hours. (B) 300 Units of caspases 1–9 were incubated with GST-lamin A for two hours. Intact and cleaved lamin A were detected via western blotting using anti-GST antibody.

    Journal: PLoS ONE

    Article Title: A Whole Cell Assay to Measure Caspase-6 Activity by Detecting Cleavage of Lamin A/C

    doi: 10.1371/journal.pone.0030376

    Figure Lengend Snippet: Western blot detection of recombinant GST-lamin A processing by purified caspases. (A) The indicated concentration of caspase-6 was incubated with GST-lamin A for two hours. (B) 300 Units of caspases 1–9 were incubated with GST-lamin A for two hours. Intact and cleaved lamin A were detected via western blotting using anti-GST antibody.

    Article Snippet: In vitro Lamin A cleavage assay Active human recombinant caspase-6 was provided by the Protein Chemistry Department at Genentech, Inc. All other active caspases were purchased from Enzo LifeSciences (Cat # BML-AK010) and used at a final concentration of 5 U/µl (300U total; see manufacturer's guide for description).

    Techniques: Western Blot, Recombinant, Purification, Concentration Assay, Incubation

    Western blot detection of lamin A/C from SKNAS neuroblastoma cells upon staurosporine treatment. (A) Schematic of the N- and C-terminal globular domains of Lamin A/C with VEID-containing central α-helical region as the site of caspase-6 proteolysis. (B) SKNAS cells were treated with DMSO control or staurosporine for 6 hours prior to cell lysis. Lysates were probed for small lamin A/C subunit (Lanes 1–2), large lamin A/C subunit (Lanes 3–4) or total lamin A/C (Lanes 5–6). (C) SKNAS cells were treated with DMSO or staurosporine for the indicated time prior to cell lysis. Lysates were probed for large lamin A/C subunit (red) or β-Actin (green).

    Journal: PLoS ONE

    Article Title: A Whole Cell Assay to Measure Caspase-6 Activity by Detecting Cleavage of Lamin A/C

    doi: 10.1371/journal.pone.0030376

    Figure Lengend Snippet: Western blot detection of lamin A/C from SKNAS neuroblastoma cells upon staurosporine treatment. (A) Schematic of the N- and C-terminal globular domains of Lamin A/C with VEID-containing central α-helical region as the site of caspase-6 proteolysis. (B) SKNAS cells were treated with DMSO control or staurosporine for 6 hours prior to cell lysis. Lysates were probed for small lamin A/C subunit (Lanes 1–2), large lamin A/C subunit (Lanes 3–4) or total lamin A/C (Lanes 5–6). (C) SKNAS cells were treated with DMSO or staurosporine for the indicated time prior to cell lysis. Lysates were probed for large lamin A/C subunit (red) or β-Actin (green).

    Article Snippet: In vitro Lamin A cleavage assay Active human recombinant caspase-6 was provided by the Protein Chemistry Department at Genentech, Inc. All other active caspases were purchased from Enzo LifeSciences (Cat # BML-AK010) and used at a final concentration of 5 U/µl (300U total; see manufacturer's guide for description).

    Techniques: Western Blot, Lysis

    Apoptosis-mediated cleavage of lamin A/C is elevated in wild-type relative to caspase-6 KO fibroblasts. Fibroblasts derived from caspase-6 KO (▪) or wild type (•) mice were treated with the indicated concentration of staurosporine for 6 hours prior to detection of the small lamin A/C cleavage product. The assay was performed in quadruplicate two times with similar results; mean and standard error of the mean are reported.

    Journal: PLoS ONE

    Article Title: A Whole Cell Assay to Measure Caspase-6 Activity by Detecting Cleavage of Lamin A/C

    doi: 10.1371/journal.pone.0030376

    Figure Lengend Snippet: Apoptosis-mediated cleavage of lamin A/C is elevated in wild-type relative to caspase-6 KO fibroblasts. Fibroblasts derived from caspase-6 KO (▪) or wild type (•) mice were treated with the indicated concentration of staurosporine for 6 hours prior to detection of the small lamin A/C cleavage product. The assay was performed in quadruplicate two times with similar results; mean and standard error of the mean are reported.

    Article Snippet: In vitro Lamin A cleavage assay Active human recombinant caspase-6 was provided by the Protein Chemistry Department at Genentech, Inc. All other active caspases were purchased from Enzo LifeSciences (Cat # BML-AK010) and used at a final concentration of 5 U/µl (300U total; see manufacturer's guide for description).

    Techniques: Derivative Assay, Mouse Assay, Concentration Assay

    Characterization of caspase cleavage site-specific antibody to N-GFAP ( A ) Caspase-cleaved GFAP was analyzed by immunoblotting with the indicated antibodies. The SMI-21 antibody recognized both intact GFAP and N-GFAP (lane 1), whereas the D225 antibody recognized N-GFAP but not intact protein (lane 2). ( B ) The D225 antibody also recognized purified recombinant N-GFAP (lane 1) but not C-GFAP ( B , lane 2), confirming the specificity of this antibody. ( C ) The presence of C-GFAP was demonstrated by immunoblotting with GA-5 antibody (lane 2). ( D ) Purified recombinant human (lane 1) and mouse GFAP (lane 3) digested with active caspase 6 (lanes 2 and 4) were analyzed by immunoblotting. Notice that the D225 antibody recognized caspase-generated N-terminal fragments from both human (lane 2) and mouse GFAP (lane 4).

    Journal: ASN NEURO

    Article Title: Caspase cleavage of GFAP produces an assembly-compromised proteolytic fragment that promotes filament aggregation

    doi: 10.1042/AN20130032

    Figure Lengend Snippet: Characterization of caspase cleavage site-specific antibody to N-GFAP ( A ) Caspase-cleaved GFAP was analyzed by immunoblotting with the indicated antibodies. The SMI-21 antibody recognized both intact GFAP and N-GFAP (lane 1), whereas the D225 antibody recognized N-GFAP but not intact protein (lane 2). ( B ) The D225 antibody also recognized purified recombinant N-GFAP (lane 1) but not C-GFAP ( B , lane 2), confirming the specificity of this antibody. ( C ) The presence of C-GFAP was demonstrated by immunoblotting with GA-5 antibody (lane 2). ( D ) Purified recombinant human (lane 1) and mouse GFAP (lane 3) digested with active caspase 6 (lanes 2 and 4) were analyzed by immunoblotting. Notice that the D225 antibody recognized caspase-generated N-terminal fragments from both human (lane 2) and mouse GFAP (lane 4).

    Article Snippet: Caspase cleavage of GFAP in vitro Purified human recombinant GFAP (1 μg) was diluted in caspase assay buffer (50 mM HEPES, pH 7.2, 50 mM NaCl, 0.1% (w/v) CHAPS, 10 mM EDTA, 10 mM DTT (dithiothreitol) and 5% (v/v) glycerol) in the absence or presence of active recombinant human caspase 3 or caspase 6 (BioVision) at a final concentration of 0.25 U/μl.

    Techniques: Purification, Recombinant, Generated

    Caspase sensitivity of AxD-causing GFAP mutant U343MG cells transiently transfected with R239H GFAP were fixed at 48 h after transfection. Cells were processed for double-label immunofluorescence microscopy using SMI-21 anti-GFAP ( A and D , green channel) and either D225 ( B , red channel) or anti-active caspase 6 ( E , red channel) antibodies. Merged image shows the region of colocalization appearing yellow ( C and F ). Notice that GFAP-rich aggregates in cells transfected with R239H GFAP ( A and D , arrows) were also immunopositive for both N-GFAP ( B , arrows) and active caspase 6 ( E , arrows). Bar, 10 μm. ( G ) Supernatant (S) and pellet (P) fractions prepared from untransfected (lanes 1 and 3) or R239H GFAP-transfected (lanes 2 and 4) cells were analyzed by immunoblotting with the SMI-21 antibody. Notice a ~26 kDa degradation product was detected in the pellet fraction of R239H-transfected cells (lane 4). (H) The identity of this fragment was confirmed by immunoprecipitating N-GFAP from the pellet fraction (lane 1) with the D225 antibody, followed by immunoblotting with the SMI-21 antibody (lane 2). (I) Immunoblotting analysis of total cell lysates with the SMI-21 antibody showed that cells transfected with R239H GFAP generated N-GFAP (lane 1, p26), whose production was inhibited by the caspase inhibitor zVAD-fmk (lane 2).

    Journal: ASN NEURO

    Article Title: Caspase cleavage of GFAP produces an assembly-compromised proteolytic fragment that promotes filament aggregation

    doi: 10.1042/AN20130032

    Figure Lengend Snippet: Caspase sensitivity of AxD-causing GFAP mutant U343MG cells transiently transfected with R239H GFAP were fixed at 48 h after transfection. Cells were processed for double-label immunofluorescence microscopy using SMI-21 anti-GFAP ( A and D , green channel) and either D225 ( B , red channel) or anti-active caspase 6 ( E , red channel) antibodies. Merged image shows the region of colocalization appearing yellow ( C and F ). Notice that GFAP-rich aggregates in cells transfected with R239H GFAP ( A and D , arrows) were also immunopositive for both N-GFAP ( B , arrows) and active caspase 6 ( E , arrows). Bar, 10 μm. ( G ) Supernatant (S) and pellet (P) fractions prepared from untransfected (lanes 1 and 3) or R239H GFAP-transfected (lanes 2 and 4) cells were analyzed by immunoblotting with the SMI-21 antibody. Notice a ~26 kDa degradation product was detected in the pellet fraction of R239H-transfected cells (lane 4). (H) The identity of this fragment was confirmed by immunoprecipitating N-GFAP from the pellet fraction (lane 1) with the D225 antibody, followed by immunoblotting with the SMI-21 antibody (lane 2). (I) Immunoblotting analysis of total cell lysates with the SMI-21 antibody showed that cells transfected with R239H GFAP generated N-GFAP (lane 1, p26), whose production was inhibited by the caspase inhibitor zVAD-fmk (lane 2).

    Article Snippet: Caspase cleavage of GFAP in vitro Purified human recombinant GFAP (1 μg) was diluted in caspase assay buffer (50 mM HEPES, pH 7.2, 50 mM NaCl, 0.1% (w/v) CHAPS, 10 mM EDTA, 10 mM DTT (dithiothreitol) and 5% (v/v) glycerol) in the absence or presence of active recombinant human caspase 3 or caspase 6 (BioVision) at a final concentration of 0.25 U/μl.

    Techniques: Mutagenesis, Transfection, Immunofluorescence, Microscopy, Generated

    GFAP is specifically cleaved at Asp 225 by caspase 6 in vitro ( A ) Purified recombinant human GFAP was either untreated (lane 1) or treated with 2.5 U of active caspase 6 (lane 2) for 1 h at 37°C. The reaction products were separated by SDS/PAGE, followed by Coomassie Blue staining. ( B ) GFAP cleaved by active caspase 6 generated two prominent proteolytic fragments, p26 and p24 (A, lane 2), which were recognized by the anti-GFAP antibodies SMI-21 (lane 1) and GA-5 (lane 2), antibodies, respectively. Both antibodies also recognized intact GFAP (lanes 1 and 2). ( C ) Caspase cleavage of Myc-GFAP generated a proteolytic fragment that was recognized by both the anti-GFAP SMI-21 (lane 2) and anti-Myc (lane 4) antibodies. ( D ) The D225E mutant GFAP was resistant to caspase 6 cleavage (lane 2), whereas caspase cleavage of wild-type GFAP generated appropriately sized proteolytic products (lane 1). The molecular mass markers (in kDa) are indicated at the left of each panel. ( E ) A schematic view of the structural organization and caspase-mediated digestion of GFAP. GFAP comprises a central α-helical rod domain, flanked by non-helical head and tail domains (denoted by black bars). Within this rod domain, subhelical segments (denoted by boxes) are connected by short linker sequences (denoted by black bars). p26 and p24 represent the major caspase-cleaved GFAP fragments that migrate on SDS/PAGE with apparent molecular masses of 26 and 24 kDa, respectively. The single-letter amino acid codes for the caspase cleavage site are also indicated. The ability of the anti-GFAP antibodies to detect specific GFAP fragments is summarized. (+), immunopositive; (−), immunonegative.

    Journal: ASN NEURO

    Article Title: Caspase cleavage of GFAP produces an assembly-compromised proteolytic fragment that promotes filament aggregation

    doi: 10.1042/AN20130032

    Figure Lengend Snippet: GFAP is specifically cleaved at Asp 225 by caspase 6 in vitro ( A ) Purified recombinant human GFAP was either untreated (lane 1) or treated with 2.5 U of active caspase 6 (lane 2) for 1 h at 37°C. The reaction products were separated by SDS/PAGE, followed by Coomassie Blue staining. ( B ) GFAP cleaved by active caspase 6 generated two prominent proteolytic fragments, p26 and p24 (A, lane 2), which were recognized by the anti-GFAP antibodies SMI-21 (lane 1) and GA-5 (lane 2), antibodies, respectively. Both antibodies also recognized intact GFAP (lanes 1 and 2). ( C ) Caspase cleavage of Myc-GFAP generated a proteolytic fragment that was recognized by both the anti-GFAP SMI-21 (lane 2) and anti-Myc (lane 4) antibodies. ( D ) The D225E mutant GFAP was resistant to caspase 6 cleavage (lane 2), whereas caspase cleavage of wild-type GFAP generated appropriately sized proteolytic products (lane 1). The molecular mass markers (in kDa) are indicated at the left of each panel. ( E ) A schematic view of the structural organization and caspase-mediated digestion of GFAP. GFAP comprises a central α-helical rod domain, flanked by non-helical head and tail domains (denoted by black bars). Within this rod domain, subhelical segments (denoted by boxes) are connected by short linker sequences (denoted by black bars). p26 and p24 represent the major caspase-cleaved GFAP fragments that migrate on SDS/PAGE with apparent molecular masses of 26 and 24 kDa, respectively. The single-letter amino acid codes for the caspase cleavage site are also indicated. The ability of the anti-GFAP antibodies to detect specific GFAP fragments is summarized. (+), immunopositive; (−), immunonegative.

    Article Snippet: Caspase cleavage of GFAP in vitro Purified human recombinant GFAP (1 μg) was diluted in caspase assay buffer (50 mM HEPES, pH 7.2, 50 mM NaCl, 0.1% (w/v) CHAPS, 10 mM EDTA, 10 mM DTT (dithiothreitol) and 5% (v/v) glycerol) in the absence or presence of active recombinant human caspase 3 or caspase 6 (BioVision) at a final concentration of 0.25 U/μl.

    Techniques: In Vitro, Purification, Recombinant, SDS Page, Staining, Generated, Mutagenesis

    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: Caspase-3, caspase-9, caspase-6 and caspase-8 antibodies were from Cell Signaling Technology (#9662, #9502, #9762, #9746); p53 antibody (DO-1) from Santa Cruz Biotechnology (#sc-126); GAPDH from Ambion (#Am4300); Alexa-488 from Invitrogen (# ); S-tag-HRP from Bethyl Laboratories (#A190-134P), and MDM2 antibody (2A10) was a kind gift from Dr. Borek Vojtesek.

    Techniques: Mutagenesis, Recombinant, Incubation, Activity Assay

    Purified antibodies specifically recognize cleaved caspase substrates with a pattern of ‘DXXD'. Apoptosis was induced by 5-FU and TRAIL in HCT116 cells as described in ‘Materials and Methods'. QVD-OPH was added to block caspase activities as indicated. Cell lysate from 10 7 cells was immuno-precipitated with 20 μ g of purified antibodies, then detected with antibodies against PARP ( a ), caspase 6 ( b ) or cleaved CK-18 ( c ), separately. The purified antibodies specifically recognize not only the cleaved PARP (DEVD) and caspase 6 (DVVD) whose caspase cleavage sites are one of the eight peptides used for immunization, but also the cleaved CK-18 (DALD), whose tetrapeptide cleavage site was not one of the eight peptides, but has aspartic acid at the P1 and P4 position. However, the purified antibodies do not recognize the cleaved caspase 7 (IQAD) or lamin A (VEID) whose tetrapeptide cleavage sites has a ‘D' at P1 but not P4 position (data not shown). Rabbit IgGs in the immune precipitate were detected as artifacts in western blots (as labeled) by the secondary antirabbit antibodies. This was due to the fact that both the NEAs and the detection antibodies (i.e. anti-PARP and anti-caspase 6) were raised in the same species (rabbit). Furthermore, to avoid precipitation, samples were not boiled before loading onto gels, therefore IgG oligmers were seen on the blots. ( d ) Purified antibodies specifically pull down cleaved recombinant CK-18. A total of 10 μ g of recombinant CK-18 was cleaved with caspase 3 in vitro . QVD-OPH was added to block caspase activities as indicated. The reaction was precipitated with purified antibodies and then detected with antibody against cleaved CK-18. ( e ) Peptides with a ‘D' at P1, or P1 and P4 (IETD, LQTD, SECD, DCRD, FRHD and FAED) were coated on the ELISA plates, and probed with purified antibodies. Scramble 3-DETD and Scramble 3 peptides were coated as positive and negative control, respectively. The antibodies only recognize the peptides with aspartic acids at both the P1 and P4 position (i.e. DCRD and DETD), but not the ones with a ‘D' only at P1 position

    Journal: Cell Death & Disease

    Article Title: Generation and characterization of antibodies specific for caspase-cleaved neo-epitopes: a novel approach

    doi: 10.1038/cddis.2011.91

    Figure Lengend Snippet: Purified antibodies specifically recognize cleaved caspase substrates with a pattern of ‘DXXD'. Apoptosis was induced by 5-FU and TRAIL in HCT116 cells as described in ‘Materials and Methods'. QVD-OPH was added to block caspase activities as indicated. Cell lysate from 10 7 cells was immuno-precipitated with 20 μ g of purified antibodies, then detected with antibodies against PARP ( a ), caspase 6 ( b ) or cleaved CK-18 ( c ), separately. The purified antibodies specifically recognize not only the cleaved PARP (DEVD) and caspase 6 (DVVD) whose caspase cleavage sites are one of the eight peptides used for immunization, but also the cleaved CK-18 (DALD), whose tetrapeptide cleavage site was not one of the eight peptides, but has aspartic acid at the P1 and P4 position. However, the purified antibodies do not recognize the cleaved caspase 7 (IQAD) or lamin A (VEID) whose tetrapeptide cleavage sites has a ‘D' at P1 but not P4 position (data not shown). Rabbit IgGs in the immune precipitate were detected as artifacts in western blots (as labeled) by the secondary antirabbit antibodies. This was due to the fact that both the NEAs and the detection antibodies (i.e. anti-PARP and anti-caspase 6) were raised in the same species (rabbit). Furthermore, to avoid precipitation, samples were not boiled before loading onto gels, therefore IgG oligmers were seen on the blots. ( d ) Purified antibodies specifically pull down cleaved recombinant CK-18. A total of 10 μ g of recombinant CK-18 was cleaved with caspase 3 in vitro . QVD-OPH was added to block caspase activities as indicated. The reaction was precipitated with purified antibodies and then detected with antibody against cleaved CK-18. ( e ) Peptides with a ‘D' at P1, or P1 and P4 (IETD, LQTD, SECD, DCRD, FRHD and FAED) were coated on the ELISA plates, and probed with purified antibodies. Scramble 3-DETD and Scramble 3 peptides were coated as positive and negative control, respectively. The antibodies only recognize the peptides with aspartic acids at both the P1 and P4 position (i.e. DCRD and DETD), but not the ones with a ‘D' only at P1 position

    Article Snippet: The following primary antibodies were used in immunoblotting: anti-PARP (Cell Signaling Technologies, Danvers, MA, USA), anti-Caspase 6 (Cell Signaling Technologies), M30 (Peviva through Diapharma, West Chester, OH, USA).

    Techniques: Purification, Blocking Assay, Western Blot, Labeling, Recombinant, In Vitro, Enzyme-linked Immunosorbent Assay, Negative Control

    Raptor cleavage by caspase-6 and other caspases. ( a ) Jurkat T-cell lysates were incubated with recombinant caspases-3, -6, -7 for 2 h. Cleavage of raptor and physiological substrates of caspases (PARP and lamin A/C) were monitored and compared with the positive control sample (Jurkat T cells treated with STS for 2 h). ( b ) The effect of z-VAD-fmk on the recombinant caspase-6-mediated cleavage of raptor in Jurkat T-cell lysate. ( c ) Cartoon of recombinant mTORC1 (composed of mLST8, mTOR (1362-end) and recombinant raptor). FAT, focal adhesion targeting; FRB, FKBP12-rapamycin binding; FATC, C-term focal adhesion targeting. ( d ) Cleavage of the full-length recombinant raptor (inside recombinant mTORC1) by recombinant caspase-6, -3 and -7 in the presence (or not) of z-VAD-fmk. ( e ) WT versus caspase-6 K.O. KBM-7 cells were treated with STS or FasL for 4 h and then cell lysates were analyzed by SDS-PAGE and western blot.

    Journal: Cell Death Discovery

    Article Title: Caspase-mediated cleavage of raptor participates in the inactivation of mTORC1 during cell death

    doi: 10.1038/cddiscovery.2016.24

    Figure Lengend Snippet: Raptor cleavage by caspase-6 and other caspases. ( a ) Jurkat T-cell lysates were incubated with recombinant caspases-3, -6, -7 for 2 h. Cleavage of raptor and physiological substrates of caspases (PARP and lamin A/C) were monitored and compared with the positive control sample (Jurkat T cells treated with STS for 2 h). ( b ) The effect of z-VAD-fmk on the recombinant caspase-6-mediated cleavage of raptor in Jurkat T-cell lysate. ( c ) Cartoon of recombinant mTORC1 (composed of mLST8, mTOR (1362-end) and recombinant raptor). FAT, focal adhesion targeting; FRB, FKBP12-rapamycin binding; FATC, C-term focal adhesion targeting. ( d ) Cleavage of the full-length recombinant raptor (inside recombinant mTORC1) by recombinant caspase-6, -3 and -7 in the presence (or not) of z-VAD-fmk. ( e ) WT versus caspase-6 K.O. KBM-7 cells were treated with STS or FasL for 4 h and then cell lysates were analyzed by SDS-PAGE and western blot.

    Article Snippet: Antibodies, reagents and drugs Anti-raptor (cat. no. 2280), anti-mTOR (cat. no. 2983), anti-phospho-mTOR Ser2448 (cat. no. 5536), anti-4E-BP1 (cat. no. 9452), anti-phospho-4E-BP1 Thr37/46 (cat. no. 2855), anti-p70 S6K (cat. no. 9202), anti-phospho-S6K Thr389 (cat. no. 9234), anti-cleaved-caspase-7 (cat. no. 9491), anti-cleaved-caspase-6 (cat. no. 9761), anti-caspase-6 (cat. no. 9762), anti-caspase-3 (cat. no. 9662), anti-PARP (cat. no. 9542), anti-lamin A/C (cat. no. 2032) and anti-IL-1β (cat. no. 12242) antibodies were purchased from Cell Signaling Technologies (Danvers, MA, USA).

    Techniques: Incubation, Recombinant, Positive Control, Binding Assay, SDS Page, Western Blot

    Cleavage of raptor at the C-term DDA DD (amino acids 939–943) residue and at the N-term DEA D LT D (amino acids 17–23) residue. ( a ) Cartoon of raptor and its putative C-term caspase-mediated cleavage residues (UniProtKB: Q8N122). The recombinant N-term raptor protein (amino acids 1–379) is represented by the arrow. Different N-term tags (HA or GST) were used according to the raptor constructs. ( b ) Jurkat T cells were transfected with GFP, WT, DDAAA or AVAAKA raptor and treated with STS for 2 h. ( c ) Jurkat T cells were transfected with GFP, WT or DDAAA raptor and treated with FasL for 2 h. ( d ) Recombinant N-term raptor was incubated with recombinant caspase-6 for 2 h in the presence (or in the absence) of z-VAD-fmk. ( e ) Recombinant raptor was incubated with recombinant caspase-6 and directly inactivated at 95 °C for 10 min or incubated for 3 h at 37 °C. The samples were analyzed by tandem MS.

    Journal: Cell Death Discovery

    Article Title: Caspase-mediated cleavage of raptor participates in the inactivation of mTORC1 during cell death

    doi: 10.1038/cddiscovery.2016.24

    Figure Lengend Snippet: Cleavage of raptor at the C-term DDA DD (amino acids 939–943) residue and at the N-term DEA D LT D (amino acids 17–23) residue. ( a ) Cartoon of raptor and its putative C-term caspase-mediated cleavage residues (UniProtKB: Q8N122). The recombinant N-term raptor protein (amino acids 1–379) is represented by the arrow. Different N-term tags (HA or GST) were used according to the raptor constructs. ( b ) Jurkat T cells were transfected with GFP, WT, DDAAA or AVAAKA raptor and treated with STS for 2 h. ( c ) Jurkat T cells were transfected with GFP, WT or DDAAA raptor and treated with FasL for 2 h. ( d ) Recombinant N-term raptor was incubated with recombinant caspase-6 for 2 h in the presence (or in the absence) of z-VAD-fmk. ( e ) Recombinant raptor was incubated with recombinant caspase-6 and directly inactivated at 95 °C for 10 min or incubated for 3 h at 37 °C. The samples were analyzed by tandem MS.

    Article Snippet: Antibodies, reagents and drugs Anti-raptor (cat. no. 2280), anti-mTOR (cat. no. 2983), anti-phospho-mTOR Ser2448 (cat. no. 5536), anti-4E-BP1 (cat. no. 9452), anti-phospho-4E-BP1 Thr37/46 (cat. no. 2855), anti-p70 S6K (cat. no. 9202), anti-phospho-S6K Thr389 (cat. no. 9234), anti-cleaved-caspase-7 (cat. no. 9491), anti-cleaved-caspase-6 (cat. no. 9761), anti-caspase-6 (cat. no. 9762), anti-caspase-3 (cat. no. 9662), anti-PARP (cat. no. 9542), anti-lamin A/C (cat. no. 2032) and anti-IL-1β (cat. no. 12242) antibodies were purchased from Cell Signaling Technologies (Danvers, MA, USA).

    Techniques: Recombinant, Construct, Transfection, Incubation, Mass Spectrometry

    In vitro cleavage of raptor by recombinant caspase-1 and -6. ( a ) Jurkat T-cell lysates were incubated with two units of recombinant caspase-1 (C1), caspase-2 (C2), caspase-3 (C3), caspase-6 (C6), caspase-7 (C7), caspase-8 (C8) or caspase-9 (C9) and raptor cleavage was monitored and compared with a STS-treated Jurkat T-cell lysate. ( b ) Time-dependant in vitro cleavage of raptor by caspase-1, -3 or -6 in Jurkat T-cell lysates using two units of each recombinant proteins.

    Journal: Cell Death Discovery

    Article Title: Caspase-mediated cleavage of raptor participates in the inactivation of mTORC1 during cell death

    doi: 10.1038/cddiscovery.2016.24

    Figure Lengend Snippet: In vitro cleavage of raptor by recombinant caspase-1 and -6. ( a ) Jurkat T-cell lysates were incubated with two units of recombinant caspase-1 (C1), caspase-2 (C2), caspase-3 (C3), caspase-6 (C6), caspase-7 (C7), caspase-8 (C8) or caspase-9 (C9) and raptor cleavage was monitored and compared with a STS-treated Jurkat T-cell lysate. ( b ) Time-dependant in vitro cleavage of raptor by caspase-1, -3 or -6 in Jurkat T-cell lysates using two units of each recombinant proteins.

    Article Snippet: Antibodies, reagents and drugs Anti-raptor (cat. no. 2280), anti-mTOR (cat. no. 2983), anti-phospho-mTOR Ser2448 (cat. no. 5536), anti-4E-BP1 (cat. no. 9452), anti-phospho-4E-BP1 Thr37/46 (cat. no. 2855), anti-p70 S6K (cat. no. 9202), anti-phospho-S6K Thr389 (cat. no. 9234), anti-cleaved-caspase-7 (cat. no. 9491), anti-cleaved-caspase-6 (cat. no. 9761), anti-caspase-6 (cat. no. 9762), anti-caspase-3 (cat. no. 9662), anti-PARP (cat. no. 9542), anti-lamin A/C (cat. no. 2032) and anti-IL-1β (cat. no. 12242) antibodies were purchased from Cell Signaling Technologies (Danvers, MA, USA).

    Techniques: In Vitro, Recombinant, Incubation

    Spiking with lamin A allows for the detection of caspase-6 activity before the cleavage of endogenous lamins. A: COS7 cells were transiently transfected with recombinant human caspase-6 and cultured for the indicated amounts of time. The active caspase-6 fragment can be observed by Western blotting 9 h after transfection and is increasingly abundant after 24 h. B: COS7 cells were transfected as in A . The cleavage of endogenous lamin A protein can be observed with the ELISA method after 24 h, whereas spiking of lysates with lamin A protein leads to a robust detection of caspase-6 activity 9 h after transfection. Statistical significance was assessed by 2-way ANOVA and post-hoc Bonferroni comparisons: *** p

    Journal: PLoS ONE

    Article Title: A Quantitative Method for the Specific Assessment of Caspase-6 Activity in Cell Culture

    doi: 10.1371/journal.pone.0027680

    Figure Lengend Snippet: Spiking with lamin A allows for the detection of caspase-6 activity before the cleavage of endogenous lamins. A: COS7 cells were transiently transfected with recombinant human caspase-6 and cultured for the indicated amounts of time. The active caspase-6 fragment can be observed by Western blotting 9 h after transfection and is increasingly abundant after 24 h. B: COS7 cells were transfected as in A . The cleavage of endogenous lamin A protein can be observed with the ELISA method after 24 h, whereas spiking of lysates with lamin A protein leads to a robust detection of caspase-6 activity 9 h after transfection. Statistical significance was assessed by 2-way ANOVA and post-hoc Bonferroni comparisons: *** p

    Article Snippet: Pure lamin A protein and lamin B1 antibody were from Abcam (cat. nos. ab8982 and ab83472), antibody against full-length caspase-6 was from Cell Signaling Technology (cat. no. 9762), antibody against caspase-3 was from Cell Signaling Technology (cat. no. 9662), antibody against actin was from Chemicon (cat. no. MAB1501R).

    Techniques: Activity Assay, Transfection, Recombinant, Cell Culture, Western Blot, Enzyme-linked Immunosorbent Assay

    VEID, but not lamin A+C, is cleaved in the absence of caspase-6. A: Caspase-6 protein (full-length, 32 kDa) is detected in MEFs generated from C6wt, but not C6ko mice. B: Endogenous lamin A protein (70 kDa) is cleaved in wt, but not C6ko MEFs after staurosporine stress for 4 h or longer. The antibody cross-reacts with full-length lamin C (60 kDa), and the cleaved band at 28 kDa has the same size for both lamin A+C (lower panel). C: MCF-7 cells express caspase-6, but not caspase-3 protein, whereas both C6wt and C6ko MEFs contain both caspases. hu: human, m: mouse, ns: non-specific band. D: MCF-7 cells were stressed with 5 µM camptothecin for different amounts of time and the cleavage of endogenous lamin A and C proteins was monitored by Western blotting with antibodies antibodies #2031 (full-length lamin A+C) and #2032 (C-terminal fragments). E: Schematic representation of lamin A and C and the caspase-6 cleavage site at AA 230. The N-terminal fragments generated by caspase-6 cleavage (red) have the same size (28 kDa) for both lamin A+C. F: C6wt or C6ko MEFs were stressed with 50 nM staurosporine for different amounts of time, lysates were generated and analyzed for cleavage of VEID-Afc. C6wt cells show a significant increase in fluorescence at each timepoint, the fluorescence signal obtained from C6ko lysates only reach a statistically significant difference from baseline after 4 h. C6ko MEFs stressed with staurosporine for 4 h or more show the same levels of VEID proteolysis as wt cells. Error bars are the SEM of N = 3 of 4 independent experiments. Statistical significance was assessed by 2-way ANOVA and post-hoc Bonferroni comparisons: *** p

    Journal: PLoS ONE

    Article Title: A Quantitative Method for the Specific Assessment of Caspase-6 Activity in Cell Culture

    doi: 10.1371/journal.pone.0027680

    Figure Lengend Snippet: VEID, but not lamin A+C, is cleaved in the absence of caspase-6. A: Caspase-6 protein (full-length, 32 kDa) is detected in MEFs generated from C6wt, but not C6ko mice. B: Endogenous lamin A protein (70 kDa) is cleaved in wt, but not C6ko MEFs after staurosporine stress for 4 h or longer. The antibody cross-reacts with full-length lamin C (60 kDa), and the cleaved band at 28 kDa has the same size for both lamin A+C (lower panel). C: MCF-7 cells express caspase-6, but not caspase-3 protein, whereas both C6wt and C6ko MEFs contain both caspases. hu: human, m: mouse, ns: non-specific band. D: MCF-7 cells were stressed with 5 µM camptothecin for different amounts of time and the cleavage of endogenous lamin A and C proteins was monitored by Western blotting with antibodies antibodies #2031 (full-length lamin A+C) and #2032 (C-terminal fragments). E: Schematic representation of lamin A and C and the caspase-6 cleavage site at AA 230. The N-terminal fragments generated by caspase-6 cleavage (red) have the same size (28 kDa) for both lamin A+C. F: C6wt or C6ko MEFs were stressed with 50 nM staurosporine for different amounts of time, lysates were generated and analyzed for cleavage of VEID-Afc. C6wt cells show a significant increase in fluorescence at each timepoint, the fluorescence signal obtained from C6ko lysates only reach a statistically significant difference from baseline after 4 h. C6ko MEFs stressed with staurosporine for 4 h or more show the same levels of VEID proteolysis as wt cells. Error bars are the SEM of N = 3 of 4 independent experiments. Statistical significance was assessed by 2-way ANOVA and post-hoc Bonferroni comparisons: *** p

    Article Snippet: Pure lamin A protein and lamin B1 antibody were from Abcam (cat. nos. ab8982 and ab83472), antibody against full-length caspase-6 was from Cell Signaling Technology (cat. no. 9762), antibody against caspase-3 was from Cell Signaling Technology (cat. no. 9662), antibody against actin was from Chemicon (cat. no. MAB1501R).

    Techniques: Generated, Mouse Assay, Western Blot, Fluorescence

    Cleavage of lamin B1 in primary neuronal cultures is not caspase-6 specific, but spiking with lamin A provides a specific readout. A: Lamins B1 and B2 differ from lamins A and C in their caspase cleavage site (VEVD versus VEID, respectively). B: Lamin B1 is cleaved after camptothecin stress in primary cortical neuronal cultures derived from C6wt and C6ko mice. C: Spiking with lamin A protein results in an increased ELISA signal in lysates from camptothecin-stressed C6wt, but not C6ko neurons. Boxes represent the 25 th –75 th percentile, whiskers represent the minimum and maximum. Data from 5 (C6ko) and 7 (C6wt) separate cultures are shown. Statistical significance was assessed by 2-way ANOVA and post-hoc Bonferroni comparisons: *** p

    Journal: PLoS ONE

    Article Title: A Quantitative Method for the Specific Assessment of Caspase-6 Activity in Cell Culture

    doi: 10.1371/journal.pone.0027680

    Figure Lengend Snippet: Cleavage of lamin B1 in primary neuronal cultures is not caspase-6 specific, but spiking with lamin A provides a specific readout. A: Lamins B1 and B2 differ from lamins A and C in their caspase cleavage site (VEVD versus VEID, respectively). B: Lamin B1 is cleaved after camptothecin stress in primary cortical neuronal cultures derived from C6wt and C6ko mice. C: Spiking with lamin A protein results in an increased ELISA signal in lysates from camptothecin-stressed C6wt, but not C6ko neurons. Boxes represent the 25 th –75 th percentile, whiskers represent the minimum and maximum. Data from 5 (C6ko) and 7 (C6wt) separate cultures are shown. Statistical significance was assessed by 2-way ANOVA and post-hoc Bonferroni comparisons: *** p

    Article Snippet: Pure lamin A protein and lamin B1 antibody were from Abcam (cat. nos. ab8982 and ab83472), antibody against full-length caspase-6 was from Cell Signaling Technology (cat. no. 9762), antibody against caspase-3 was from Cell Signaling Technology (cat. no. 9662), antibody against actin was from Chemicon (cat. no. MAB1501R).

    Techniques: Derivative Assay, Mouse Assay, Enzyme-linked Immunosorbent Assay

    The lamin A protein is a more specific substrate than its VEID peptide. A: 100 µM VEID-Afc was incubated with different amounts of caspase -3, -6 or -7 for 1 h at 37°C. Fluorescence generated by cleavage was monitored over time, and the initial, linear portion of the curve was used to calculate the reaction velocity. VEID is preferentially cleaved by caspase-6, but also cross-reacts with caspases -3 and -7 at higher concentrations. Error bars are the SEM of N≥3 of 3 independent experiments. B: 5 µM VEID-Afc was incubated with 0.5 µM caspase-6 or different amounts of caspase-3 for 1 h at 37°C. The reaction velocity was calculated as in A. Even at this low concentration of VEID-Afc, the peptide substrate can be cleaved by caspase-3, and an 8 fold higher molar concentration of caspase-3 than caspase-6 results in a higher signal for VEID cleavage by caspase-3 than caspase-6. Error bars are the SEM of N = 3 independent experiments, statistical significance was assessed by 1-way ANOVA and post-hoc Dunnett comparisons: *** p

    Journal: PLoS ONE

    Article Title: A Quantitative Method for the Specific Assessment of Caspase-6 Activity in Cell Culture

    doi: 10.1371/journal.pone.0027680

    Figure Lengend Snippet: The lamin A protein is a more specific substrate than its VEID peptide. A: 100 µM VEID-Afc was incubated with different amounts of caspase -3, -6 or -7 for 1 h at 37°C. Fluorescence generated by cleavage was monitored over time, and the initial, linear portion of the curve was used to calculate the reaction velocity. VEID is preferentially cleaved by caspase-6, but also cross-reacts with caspases -3 and -7 at higher concentrations. Error bars are the SEM of N≥3 of 3 independent experiments. B: 5 µM VEID-Afc was incubated with 0.5 µM caspase-6 or different amounts of caspase-3 for 1 h at 37°C. The reaction velocity was calculated as in A. Even at this low concentration of VEID-Afc, the peptide substrate can be cleaved by caspase-3, and an 8 fold higher molar concentration of caspase-3 than caspase-6 results in a higher signal for VEID cleavage by caspase-3 than caspase-6. Error bars are the SEM of N = 3 independent experiments, statistical significance was assessed by 1-way ANOVA and post-hoc Dunnett comparisons: *** p

    Article Snippet: Pure lamin A protein and lamin B1 antibody were from Abcam (cat. nos. ab8982 and ab83472), antibody against full-length caspase-6 was from Cell Signaling Technology (cat. no. 9762), antibody against caspase-3 was from Cell Signaling Technology (cat. no. 9662), antibody against actin was from Chemicon (cat. no. MAB1501R).

    Techniques: Incubation, Fluorescence, Generated, Concentration Assay

    Caspase-6 activity can be quantified by immunofluorescent staining for cleaved lamin A. A: C6wt and C6ko MEFs were stressed with camptothecin, stained with an antibody against cleaved lamin A and analyzed on an automated imaging platform. Nuclei were counterstained with DAPI, and identified by the software (blue circles). Debris (red circles) was not analyzed. The signal intensity in the cleaved lamin A channel was quantified in a ring around the nucleus (green circles). B: Quantitation of the perinuclear staining from cleaved lamin A is graphed. Untreated C6wt MEFs were normalized to 100% and the fold change in stressed C6wt, stressed and non-stressed C6ko MEFs are compared. Error bars are the SEM of N≥3 of 3 independent experiments. Statistical significance was assessed by 2-way ANOVA and post-hoc Bonferroni comparisons: *** p

    Journal: PLoS ONE

    Article Title: A Quantitative Method for the Specific Assessment of Caspase-6 Activity in Cell Culture

    doi: 10.1371/journal.pone.0027680

    Figure Lengend Snippet: Caspase-6 activity can be quantified by immunofluorescent staining for cleaved lamin A. A: C6wt and C6ko MEFs were stressed with camptothecin, stained with an antibody against cleaved lamin A and analyzed on an automated imaging platform. Nuclei were counterstained with DAPI, and identified by the software (blue circles). Debris (red circles) was not analyzed. The signal intensity in the cleaved lamin A channel was quantified in a ring around the nucleus (green circles). B: Quantitation of the perinuclear staining from cleaved lamin A is graphed. Untreated C6wt MEFs were normalized to 100% and the fold change in stressed C6wt, stressed and non-stressed C6ko MEFs are compared. Error bars are the SEM of N≥3 of 3 independent experiments. Statistical significance was assessed by 2-way ANOVA and post-hoc Bonferroni comparisons: *** p

    Article Snippet: Pure lamin A protein and lamin B1 antibody were from Abcam (cat. nos. ab8982 and ab83472), antibody against full-length caspase-6 was from Cell Signaling Technology (cat. no. 9762), antibody against caspase-3 was from Cell Signaling Technology (cat. no. 9662), antibody against actin was from Chemicon (cat. no. MAB1501R).

    Techniques: Activity Assay, Staining, Imaging, Software, Quantitation Assay

    Increased sensitivity and highly improved specificity in a lamin-based caspase-6 activity assay. A+B: Quantities of down to 10 nM active caspase-6 can be detected with the novel lamin A-based caspase-6 assay while maintaining a signal-noise ratio of > 3. C: The peptide inhibitor VEID-CHO shows a similar IC 50 value in both the VEID- and the lamin A- based caspase-6 assays. D: The lamin A-based caspase-6 assay is highly specific, with no signal generated from C6ko cells even after 6 h of staurosporine stress. Error bars are the SEM of N≥3 of 3 independent experiments. Statistical significance was assessed by 2-way ANOVA and post-hoc Bonferroni comparisons: *** p

    Journal: PLoS ONE

    Article Title: A Quantitative Method for the Specific Assessment of Caspase-6 Activity in Cell Culture

    doi: 10.1371/journal.pone.0027680

    Figure Lengend Snippet: Increased sensitivity and highly improved specificity in a lamin-based caspase-6 activity assay. A+B: Quantities of down to 10 nM active caspase-6 can be detected with the novel lamin A-based caspase-6 assay while maintaining a signal-noise ratio of > 3. C: The peptide inhibitor VEID-CHO shows a similar IC 50 value in both the VEID- and the lamin A- based caspase-6 assays. D: The lamin A-based caspase-6 assay is highly specific, with no signal generated from C6ko cells even after 6 h of staurosporine stress. Error bars are the SEM of N≥3 of 3 independent experiments. Statistical significance was assessed by 2-way ANOVA and post-hoc Bonferroni comparisons: *** p

    Article Snippet: Pure lamin A protein and lamin B1 antibody were from Abcam (cat. nos. ab8982 and ab83472), antibody against full-length caspase-6 was from Cell Signaling Technology (cat. no. 9762), antibody against caspase-3 was from Cell Signaling Technology (cat. no. 9662), antibody against actin was from Chemicon (cat. no. MAB1501R).

    Techniques: Activity Assay, Generated

    Astrocytes are resistant to caspase-6, -7, and -8 but undergo apoptosis with caspase-3. Astrocytes were injected with 20 or 100 pg/cell. Apoptosis was determined by TUNEL. Data represents the mean and SD of two experiments from three astrocyte preparations.

    Journal: The Journal of Neuroscience

    Article Title: Selective and Protracted Apoptosis in Human Primary Neurons Microinjected with Active Caspase-3, -6, -7, and -8

    doi: 10.1523/JNEUROSCI.20-22-08384.2000

    Figure Lengend Snippet: Astrocytes are resistant to caspase-6, -7, and -8 but undergo apoptosis with caspase-3. Astrocytes were injected with 20 or 100 pg/cell. Apoptosis was determined by TUNEL. Data represents the mean and SD of two experiments from three astrocyte preparations.

    Article Snippet: To determine whether other CNS cell types are vulnerable to caspase-6 or other caspases, human astrocytes were microinjected with 20 and 100 pg/cell recombinant caspase-3, -6, -7, and -8 (Fig. ).

    Techniques: Injection, TUNEL Assay

    Sublethal dose of caspase-6 renders neurons vulnerable to normally nonlethal doses of oxidative stress. Neurons were microinjected with DTR in the absence or presence of 0.1 pg/cell R-Csp-6 and submitted to serum deprivation or 0.1 μ m H 2 O 2 . Cells

    Journal: The Journal of Neuroscience

    Article Title: Selective and Protracted Apoptosis in Human Primary Neurons Microinjected with Active Caspase-3, -6, -7, and -8

    doi: 10.1523/JNEUROSCI.20-22-08384.2000

    Figure Lengend Snippet: Sublethal dose of caspase-6 renders neurons vulnerable to normally nonlethal doses of oxidative stress. Neurons were microinjected with DTR in the absence or presence of 0.1 pg/cell R-Csp-6 and submitted to serum deprivation or 0.1 μ m H 2 O 2 . Cells

    Article Snippet: To determine whether other CNS cell types are vulnerable to caspase-6 or other caspases, human astrocytes were microinjected with 20 and 100 pg/cell recombinant caspase-3, -6, -7, and -8 (Fig. ).

    Techniques: