rabbit anti ripk3  (ProSci Incorporated)


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    ProSci Incorporated rabbit anti ripk3
    A , B U937 cell lines of various genotypes expressing an inducible MLKL(1-180)-gyrase fusion were treated with doxycycline (to induce expression) and/or coumermycin (to induce dimerisation), or left untreated. After 24 h ( A ) or 48 h ( B ), cells were harvested and assessed for cell death (PI uptake measured using flow cytometry), counting a minimum of 5000 cells. Data represent three independent experiments, with the exception of the BAX −/− BAK −/− cell lines, where n = 2. Statistics were calculated in GraphPad Prism 8, and p values are shown where <0.05. C The same U937 cells used in panel A and B were treated with doxycycline alone and left overnight. After approximately 16 h, cells were harvested and lysed for analysis of fusion protein expression via western blot. The upper MLKL band (*) represents endogenous MLKL, and the lower band (arrow) represents the MLKL (1-180) -gyrase fusion protein. D Wild-type U937 were treated with TNF (100 ng/ml), Smac-mimetic (0.5 µM), and caspase inhibitor IDN-6556 (emricasan; 5 µM) (TSI) to induce necroptosis or TNF and Smac-mimetic (TS) alone to induce apoptosis. In parallel, MLKL −/− U937 expressing an inducible MLKL (1-180) -gyrase fusion were treated with doxycycline and coumermycin in the presence or absence of necrosulfonamide (NSA). Images were taken at the specified timepoints using an IncuCyte S3 System to track morphological changes and PI uptake (shown by red). Cells with a necroptotic phenotype (cellular swelling) are indicate with yellow arrows. Three fields were examined per well with representative data shown. E Wild-type U937 cells were treated with doxycycline (20 nM) to induce expression of human wild-type <t>RIPK3</t> in the presence of various cell death inhibitors, as indicated, for 24 h. At the conclusion of the experiment, cells were analysed for PI uptake using flow cytometry, with a minimum of 5000 cells counted. Data represent four or more independent experiments. F Wild-type, MLKL −/− and RIPK1 −/− U937 expressing inducible human wild-type RIPK3 were treated with doxycycline (20 nM) and IDN-6556 (5 µM), doxycycline alone, or left untreated. After 24 h, cell death was measured as PI uptake using flow cytometry. Data are a summary of six independent experiments.
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    1) Product Images from "The Lck inhibitor, AMG-47a, blocks necroptosis and implicates RIPK1 in signalling downstream of MLKL"

    Article Title: The Lck inhibitor, AMG-47a, blocks necroptosis and implicates RIPK1 in signalling downstream of MLKL

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-022-04740-w

    A , B U937 cell lines of various genotypes expressing an inducible MLKL(1-180)-gyrase fusion were treated with doxycycline (to induce expression) and/or coumermycin (to induce dimerisation), or left untreated. After 24 h ( A ) or 48 h ( B ), cells were harvested and assessed for cell death (PI uptake measured using flow cytometry), counting a minimum of 5000 cells. Data represent three independent experiments, with the exception of the BAX −/− BAK −/− cell lines, where n = 2. Statistics were calculated in GraphPad Prism 8, and p values are shown where <0.05. C The same U937 cells used in panel A and B were treated with doxycycline alone and left overnight. After approximately 16 h, cells were harvested and lysed for analysis of fusion protein expression via western blot. The upper MLKL band (*) represents endogenous MLKL, and the lower band (arrow) represents the MLKL (1-180) -gyrase fusion protein. D Wild-type U937 were treated with TNF (100 ng/ml), Smac-mimetic (0.5 µM), and caspase inhibitor IDN-6556 (emricasan; 5 µM) (TSI) to induce necroptosis or TNF and Smac-mimetic (TS) alone to induce apoptosis. In parallel, MLKL −/− U937 expressing an inducible MLKL (1-180) -gyrase fusion were treated with doxycycline and coumermycin in the presence or absence of necrosulfonamide (NSA). Images were taken at the specified timepoints using an IncuCyte S3 System to track morphological changes and PI uptake (shown by red). Cells with a necroptotic phenotype (cellular swelling) are indicate with yellow arrows. Three fields were examined per well with representative data shown. E Wild-type U937 cells were treated with doxycycline (20 nM) to induce expression of human wild-type RIPK3 in the presence of various cell death inhibitors, as indicated, for 24 h. At the conclusion of the experiment, cells were analysed for PI uptake using flow cytometry, with a minimum of 5000 cells counted. Data represent four or more independent experiments. F Wild-type, MLKL −/− and RIPK1 −/− U937 expressing inducible human wild-type RIPK3 were treated with doxycycline (20 nM) and IDN-6556 (5 µM), doxycycline alone, or left untreated. After 24 h, cell death was measured as PI uptake using flow cytometry. Data are a summary of six independent experiments.
    Figure Legend Snippet: A , B U937 cell lines of various genotypes expressing an inducible MLKL(1-180)-gyrase fusion were treated with doxycycline (to induce expression) and/or coumermycin (to induce dimerisation), or left untreated. After 24 h ( A ) or 48 h ( B ), cells were harvested and assessed for cell death (PI uptake measured using flow cytometry), counting a minimum of 5000 cells. Data represent three independent experiments, with the exception of the BAX −/− BAK −/− cell lines, where n = 2. Statistics were calculated in GraphPad Prism 8, and p values are shown where <0.05. C The same U937 cells used in panel A and B were treated with doxycycline alone and left overnight. After approximately 16 h, cells were harvested and lysed for analysis of fusion protein expression via western blot. The upper MLKL band (*) represents endogenous MLKL, and the lower band (arrow) represents the MLKL (1-180) -gyrase fusion protein. D Wild-type U937 were treated with TNF (100 ng/ml), Smac-mimetic (0.5 µM), and caspase inhibitor IDN-6556 (emricasan; 5 µM) (TSI) to induce necroptosis or TNF and Smac-mimetic (TS) alone to induce apoptosis. In parallel, MLKL −/− U937 expressing an inducible MLKL (1-180) -gyrase fusion were treated with doxycycline and coumermycin in the presence or absence of necrosulfonamide (NSA). Images were taken at the specified timepoints using an IncuCyte S3 System to track morphological changes and PI uptake (shown by red). Cells with a necroptotic phenotype (cellular swelling) are indicate with yellow arrows. Three fields were examined per well with representative data shown. E Wild-type U937 cells were treated with doxycycline (20 nM) to induce expression of human wild-type RIPK3 in the presence of various cell death inhibitors, as indicated, for 24 h. At the conclusion of the experiment, cells were analysed for PI uptake using flow cytometry, with a minimum of 5000 cells counted. Data represent four or more independent experiments. F Wild-type, MLKL −/− and RIPK1 −/− U937 expressing inducible human wild-type RIPK3 were treated with doxycycline (20 nM) and IDN-6556 (5 µM), doxycycline alone, or left untreated. After 24 h, cell death was measured as PI uptake using flow cytometry. Data are a summary of six independent experiments.

    Techniques Used: Expressing, Flow Cytometry, Western Blot

    A Three different experimental protocols were used to assess the ability of AMG-47a to inhibit cell death caused by the expression and dimerisation of the MLKL (1-180) -gyrase fusion protein. In protocol 1, inhibitors (or DMSO) were added first, followed by doxycycline and coumermycin together, so that the fusion protein could dimerise on expression. In protocol 2, addition of coumermycin was delayed to allow levels of the fusion protein to accumulate before dimerisation was induced. In protocol 3, the addition of inhibitors (or DMSO) was also delayed, so that it was added after the fusion protein had been expressed for 16 h, but before the addition of coumermycin. In all experimental methods, cells were harvested 24 h after the experiment was initiated. B Wild-type U937 cells expressing the MLKL (1-180) -gyrase fusion protein were treated using either protocol 1, 2, or 3, as described above ( A ). At the conclusion of the experiment, cells were analysed for PI uptake using flow cytometry, with a minimum of 5000 cells counted. Data represent three independent experiments; bars indicate the mean and error bars indicate standard error of the mean. Statistics were calculated in GraphPad Prism8, and p values are shown where <0.05. C Wild-type U937 cells with inducible human RIPK3 were treated with increasing doses of AMG-47a in the presence of 40 nM doxycycline, to induce expression, and 5 µM IDN-6556, to block caspase-dependent cell death. After 48 h, cells were harvested and viability assessed using the Cell-Titer Glo 2 (Promega) system. Data represent two independent experiments (four replicates per experiment) and have been normalised against doxycycline plus IDN-6556 (0% viability) and doxycycline plus IDN-6556 plus NSA (100% viability).
    Figure Legend Snippet: A Three different experimental protocols were used to assess the ability of AMG-47a to inhibit cell death caused by the expression and dimerisation of the MLKL (1-180) -gyrase fusion protein. In protocol 1, inhibitors (or DMSO) were added first, followed by doxycycline and coumermycin together, so that the fusion protein could dimerise on expression. In protocol 2, addition of coumermycin was delayed to allow levels of the fusion protein to accumulate before dimerisation was induced. In protocol 3, the addition of inhibitors (or DMSO) was also delayed, so that it was added after the fusion protein had been expressed for 16 h, but before the addition of coumermycin. In all experimental methods, cells were harvested 24 h after the experiment was initiated. B Wild-type U937 cells expressing the MLKL (1-180) -gyrase fusion protein were treated using either protocol 1, 2, or 3, as described above ( A ). At the conclusion of the experiment, cells were analysed for PI uptake using flow cytometry, with a minimum of 5000 cells counted. Data represent three independent experiments; bars indicate the mean and error bars indicate standard error of the mean. Statistics were calculated in GraphPad Prism8, and p values are shown where <0.05. C Wild-type U937 cells with inducible human RIPK3 were treated with increasing doses of AMG-47a in the presence of 40 nM doxycycline, to induce expression, and 5 µM IDN-6556, to block caspase-dependent cell death. After 48 h, cells were harvested and viability assessed using the Cell-Titer Glo 2 (Promega) system. Data represent two independent experiments (four replicates per experiment) and have been normalised against doxycycline plus IDN-6556 (0% viability) and doxycycline plus IDN-6556 plus NSA (100% viability).

    Techniques Used: Expressing, Flow Cytometry, Blocking Assay

    A, B, C U937, HT29 and MDF cell lines were treated with DMSO, AMG-47a (1 µM for U937 and MDF; 2 µM for HT29), TSI, or a combination of AMG-47a and TSI over a time course as indicated in the individual figure panels. At the conclusion of the experiment, cells were harvested and lysed for western blot analysis. All cell lines were assessed for phosphorylation and total protein of the three key necroptosis effector proteins, RIPK1, RIPK3, and MLKL, and total β-actin was used as a loading control. Where multiple bands are present, the specific band of interest is indicated with a red arrow. Data are representative of at least three independent experiments. U937 ( D ) or MDF ( E ) cell lines were treated with AMG-47a at 10 µM, the RIPK1 inhibitors (GSK-481, Nec-1s) or a RIPK3 inhibitor (GSK-872) at 20 µM, or an equivalent amount of DMSO, for 1 h at 37 °C. Cell suspensions were then heated over an increasing temperature gradient (as indicated in the methods) for 3 min. Cells were then lysed, and the amount of remaining soluble protein at each temperature point was analysed using Western blot. Results are representative of two (RIPK1 and RIPK3 in U937) or three (all other analyses) independent experiments. AMG-47a binding to RIPK1 ( F ) and RIPK3 ( G ) was measured in competitive binding assays using the KINOMEscan ® Assay Platform (DiscoverX). Graphs were plotted using the raw data supplied from DiscoverX, and a curve was fitted using non-linear regression (GraphPad Prism) to determine the K d and associated error (95% confidence interval). Data represent one (RIPK3) or two (RIPK1) independent experiments, with two runs performed in all experiments and each colour on the graph representing a separate run. All data points were used for the non-linear regression. K d determinations for individual replicates can be found in supplementary data (Supplementary Fig. ). H , I AMG-47a was serially diluted from a maximum concentration of 50 µM (RIPK1) or 100 µM (RIPK3), and its ability to inhibit human RIPK1 or RIPK3 kinase activity was assessed using the ADP-Glo Kinase Assay (Promega). The reaction was allowed to proceed for 4 h before readouts were performed, and data were normalised to DMSO (0% inhibition) and either 1 µM GSK-481 (100% inhibition of RIPK1) or 1 µM GSK-872 (100% inhibition of RIPK3). Data are a summary of two independent experiments performed in duplicate, and the IC 50 and associated error (95% confidence interval) were determined using non-linear regression, as above. Each colour on the graph represents one independent experiment.
    Figure Legend Snippet: A, B, C U937, HT29 and MDF cell lines were treated with DMSO, AMG-47a (1 µM for U937 and MDF; 2 µM for HT29), TSI, or a combination of AMG-47a and TSI over a time course as indicated in the individual figure panels. At the conclusion of the experiment, cells were harvested and lysed for western blot analysis. All cell lines were assessed for phosphorylation and total protein of the three key necroptosis effector proteins, RIPK1, RIPK3, and MLKL, and total β-actin was used as a loading control. Where multiple bands are present, the specific band of interest is indicated with a red arrow. Data are representative of at least three independent experiments. U937 ( D ) or MDF ( E ) cell lines were treated with AMG-47a at 10 µM, the RIPK1 inhibitors (GSK-481, Nec-1s) or a RIPK3 inhibitor (GSK-872) at 20 µM, or an equivalent amount of DMSO, for 1 h at 37 °C. Cell suspensions were then heated over an increasing temperature gradient (as indicated in the methods) for 3 min. Cells were then lysed, and the amount of remaining soluble protein at each temperature point was analysed using Western blot. Results are representative of two (RIPK1 and RIPK3 in U937) or three (all other analyses) independent experiments. AMG-47a binding to RIPK1 ( F ) and RIPK3 ( G ) was measured in competitive binding assays using the KINOMEscan ® Assay Platform (DiscoverX). Graphs were plotted using the raw data supplied from DiscoverX, and a curve was fitted using non-linear regression (GraphPad Prism) to determine the K d and associated error (95% confidence interval). Data represent one (RIPK3) or two (RIPK1) independent experiments, with two runs performed in all experiments and each colour on the graph representing a separate run. All data points were used for the non-linear regression. K d determinations for individual replicates can be found in supplementary data (Supplementary Fig. ). H , I AMG-47a was serially diluted from a maximum concentration of 50 µM (RIPK1) or 100 µM (RIPK3), and its ability to inhibit human RIPK1 or RIPK3 kinase activity was assessed using the ADP-Glo Kinase Assay (Promega). The reaction was allowed to proceed for 4 h before readouts were performed, and data were normalised to DMSO (0% inhibition) and either 1 µM GSK-481 (100% inhibition of RIPK1) or 1 µM GSK-872 (100% inhibition of RIPK3). Data are a summary of two independent experiments performed in duplicate, and the IC 50 and associated error (95% confidence interval) were determined using non-linear regression, as above. Each colour on the graph represents one independent experiment.

    Techniques Used: Western Blot, Binding Assay, Concentration Assay, Activity Assay, Kinase Assay, Inhibition

    A The cell death induced by forced dimerisation of the human MLKL NTD involves two distinct processes. In the first, MLKL can self-associate into discrete oligomers on addition of doxycycline. In the second, addition of coumermycin promotes the assembly of large multimeric complexes. AMG-47a is unable to inhibit the second process, thus we propose it prevents membrane attachment, translocation, or oligomerisation of the human MLKL NTD through its activity as a kinase inhibitor. As AMG-47a binds RIPK1, and as RIPK1 is required for this form of cell death, we propose the primary target of AMG-47a is RIPK1 in this context. B When a cell receives a necroptotic stimulus, a number of key events occur, beginning with assembly of a necrosome containing RIPK1, RIPK3, and MLKL. This triggers a number of events, beginning with the phosphorylation of MLKL by RIPK3, which enables a conformational change in MLKL that allows oligomerisation, translocation to cellular membranes, and ultimately cell death. Our data suggest an additional role for RIPK1 in human necroptosis by supporting the conformational change, oligomerisation, or membrane translocation of MLKL following phosphorylation by RIPK3.
    Figure Legend Snippet: A The cell death induced by forced dimerisation of the human MLKL NTD involves two distinct processes. In the first, MLKL can self-associate into discrete oligomers on addition of doxycycline. In the second, addition of coumermycin promotes the assembly of large multimeric complexes. AMG-47a is unable to inhibit the second process, thus we propose it prevents membrane attachment, translocation, or oligomerisation of the human MLKL NTD through its activity as a kinase inhibitor. As AMG-47a binds RIPK1, and as RIPK1 is required for this form of cell death, we propose the primary target of AMG-47a is RIPK1 in this context. B When a cell receives a necroptotic stimulus, a number of key events occur, beginning with assembly of a necrosome containing RIPK1, RIPK3, and MLKL. This triggers a number of events, beginning with the phosphorylation of MLKL by RIPK3, which enables a conformational change in MLKL that allows oligomerisation, translocation to cellular membranes, and ultimately cell death. Our data suggest an additional role for RIPK1 in human necroptosis by supporting the conformational change, oligomerisation, or membrane translocation of MLKL following phosphorylation by RIPK3.

    Techniques Used: Translocation Assay, Activity Assay

    rabbit anti ripk3  (ProSci Incorporated)


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    ProSci Incorporated rabbit anti ripk3
    ( A ) Schematic of the necroptosis pathway. TNF (T) activates TNFR1, the Smac-mimetic Compound A (S) blocks cIAP activity and the pan-caspase inhibitor Q-VD-OPh (Q) blocks caspase-8 activity. This TSQ stimulus results in activation of RIPK1 and <t>RIPK3,</t> and subsequent phosphorylation and activation of MLKL, which causes MLKL-mediated membrane disruption and cell death. ( B ) Schematic of the constitutively activated mouse MLKL mutant, Q343A. Expression of MLKL Q343A using doxycycline causes cell death in the absence of upstream necroptotic stimuli. This enabled a cell-based phenotypic screen for small molecules that modulate necroptosis at the level or downstream of MLKL activation. The skull and crossbones image (Mycomorphbox_Deadly.png; by Sven Manguard) in ( A , B ) was used under a Creative Commons Attribution-Share Alike 4.0 license. ( C ) Schematic of the cell-based phenotypic screen. A total of 5632 compounds from the WEHI small molecule library along with 40 kinase inhibitors were screened against wild-type or Mlkl −/− mouse dermal fibroblast (MDF) cells expressing the MLKL Q343A mutant. The ability of the small molecules to inhibit cell death was measured by CellTiter-Glo cell viability assays. ABT-869, a VEGF and PDGF receptor tyrosine kinase inhibitor, was identified as a hit. See also Supplementary Figure S1A. ( D ) Chemical structure of ABT-869 and its analogue WEHI-615. ( E ) Wild-type mouse dermal fibroblast (MDF) cells expressing the doxycycline-inducible MLKL Q343A mutant to trigger constitutive necroptosis were treated with DMSO alone, doxycycline (Dox; 1 µg/ml) and DMSO, or Dox and ABT-869 (1 µM). Cell viability was quantified by CellTiter-Glo. Data represent the mean of ≥2 technical replicates from a single experiment, with individual data points shown. See also Supplementary Figure S1A. ( F ) Mlkl −/− mouse dermal fibroblast (MDF) cells expressing the doxycycline-inducible MLKL Q343A mutant to trigger constitutive necroptosis were treated with DMSO alone, doxycycline (Dox; 1 µg/ml) and DMSO, or Dox and ABT-869 (1 µM). Cell viability was quantified by CellTiter-Glo. Data represent the mean of ≥2 technical replicates from a single experiment, with individual data points shown. See also Supplementary Figure S1A. ( G ) Wild-type mouse dermal fibroblast (MDF) cells were stimulated with TSQ (TNF, Smac-mimetic, Q-VD-OPh) to induce necroptosis and treated with increasing concentrations of ABT-869 or WEHI-615. Cell death was quantified by propidium iodide (PI) staining using flow cytometry. Data represent the mean of n = 4 independent experiments and errors bars represent SEM.
    Rabbit Anti Ripk3, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "The VEGFR/PDGFR tyrosine kinase inhibitor, ABT-869, blocks necroptosis by targeting RIPK1 kinase"

    Article Title: The VEGFR/PDGFR tyrosine kinase inhibitor, ABT-869, blocks necroptosis by targeting RIPK1 kinase

    Journal: Biochemical Journal

    doi: 10.1042/BCJ20230035

    ( A ) Schematic of the necroptosis pathway. TNF (T) activates TNFR1, the Smac-mimetic Compound A (S) blocks cIAP activity and the pan-caspase inhibitor Q-VD-OPh (Q) blocks caspase-8 activity. This TSQ stimulus results in activation of RIPK1 and RIPK3, and subsequent phosphorylation and activation of MLKL, which causes MLKL-mediated membrane disruption and cell death. ( B ) Schematic of the constitutively activated mouse MLKL mutant, Q343A. Expression of MLKL Q343A using doxycycline causes cell death in the absence of upstream necroptotic stimuli. This enabled a cell-based phenotypic screen for small molecules that modulate necroptosis at the level or downstream of MLKL activation. The skull and crossbones image (Mycomorphbox_Deadly.png; by Sven Manguard) in ( A , B ) was used under a Creative Commons Attribution-Share Alike 4.0 license. ( C ) Schematic of the cell-based phenotypic screen. A total of 5632 compounds from the WEHI small molecule library along with 40 kinase inhibitors were screened against wild-type or Mlkl −/− mouse dermal fibroblast (MDF) cells expressing the MLKL Q343A mutant. The ability of the small molecules to inhibit cell death was measured by CellTiter-Glo cell viability assays. ABT-869, a VEGF and PDGF receptor tyrosine kinase inhibitor, was identified as a hit. See also Supplementary Figure S1A. ( D ) Chemical structure of ABT-869 and its analogue WEHI-615. ( E ) Wild-type mouse dermal fibroblast (MDF) cells expressing the doxycycline-inducible MLKL Q343A mutant to trigger constitutive necroptosis were treated with DMSO alone, doxycycline (Dox; 1 µg/ml) and DMSO, or Dox and ABT-869 (1 µM). Cell viability was quantified by CellTiter-Glo. Data represent the mean of ≥2 technical replicates from a single experiment, with individual data points shown. See also Supplementary Figure S1A. ( F ) Mlkl −/− mouse dermal fibroblast (MDF) cells expressing the doxycycline-inducible MLKL Q343A mutant to trigger constitutive necroptosis were treated with DMSO alone, doxycycline (Dox; 1 µg/ml) and DMSO, or Dox and ABT-869 (1 µM). Cell viability was quantified by CellTiter-Glo. Data represent the mean of ≥2 technical replicates from a single experiment, with individual data points shown. See also Supplementary Figure S1A. ( G ) Wild-type mouse dermal fibroblast (MDF) cells were stimulated with TSQ (TNF, Smac-mimetic, Q-VD-OPh) to induce necroptosis and treated with increasing concentrations of ABT-869 or WEHI-615. Cell death was quantified by propidium iodide (PI) staining using flow cytometry. Data represent the mean of n = 4 independent experiments and errors bars represent SEM.
    Figure Legend Snippet: ( A ) Schematic of the necroptosis pathway. TNF (T) activates TNFR1, the Smac-mimetic Compound A (S) blocks cIAP activity and the pan-caspase inhibitor Q-VD-OPh (Q) blocks caspase-8 activity. This TSQ stimulus results in activation of RIPK1 and RIPK3, and subsequent phosphorylation and activation of MLKL, which causes MLKL-mediated membrane disruption and cell death. ( B ) Schematic of the constitutively activated mouse MLKL mutant, Q343A. Expression of MLKL Q343A using doxycycline causes cell death in the absence of upstream necroptotic stimuli. This enabled a cell-based phenotypic screen for small molecules that modulate necroptosis at the level or downstream of MLKL activation. The skull and crossbones image (Mycomorphbox_Deadly.png; by Sven Manguard) in ( A , B ) was used under a Creative Commons Attribution-Share Alike 4.0 license. ( C ) Schematic of the cell-based phenotypic screen. A total of 5632 compounds from the WEHI small molecule library along with 40 kinase inhibitors were screened against wild-type or Mlkl −/− mouse dermal fibroblast (MDF) cells expressing the MLKL Q343A mutant. The ability of the small molecules to inhibit cell death was measured by CellTiter-Glo cell viability assays. ABT-869, a VEGF and PDGF receptor tyrosine kinase inhibitor, was identified as a hit. See also Supplementary Figure S1A. ( D ) Chemical structure of ABT-869 and its analogue WEHI-615. ( E ) Wild-type mouse dermal fibroblast (MDF) cells expressing the doxycycline-inducible MLKL Q343A mutant to trigger constitutive necroptosis were treated with DMSO alone, doxycycline (Dox; 1 µg/ml) and DMSO, or Dox and ABT-869 (1 µM). Cell viability was quantified by CellTiter-Glo. Data represent the mean of ≥2 technical replicates from a single experiment, with individual data points shown. See also Supplementary Figure S1A. ( F ) Mlkl −/− mouse dermal fibroblast (MDF) cells expressing the doxycycline-inducible MLKL Q343A mutant to trigger constitutive necroptosis were treated with DMSO alone, doxycycline (Dox; 1 µg/ml) and DMSO, or Dox and ABT-869 (1 µM). Cell viability was quantified by CellTiter-Glo. Data represent the mean of ≥2 technical replicates from a single experiment, with individual data points shown. See also Supplementary Figure S1A. ( G ) Wild-type mouse dermal fibroblast (MDF) cells were stimulated with TSQ (TNF, Smac-mimetic, Q-VD-OPh) to induce necroptosis and treated with increasing concentrations of ABT-869 or WEHI-615. Cell death was quantified by propidium iodide (PI) staining using flow cytometry. Data represent the mean of n = 4 independent experiments and errors bars represent SEM.

    Techniques Used: Activity Assay, Activation Assay, Mutagenesis, Expressing, Staining, Flow Cytometry

    ( A , B ) Wild-type mouse dermal fibroblast (MDF) cells were treated with increasing concentrations of ABT-869 or control compounds, RIPK3 inhibitors GSK′872 and GSK′843, DMSO alone or left untreated (UT) for 1 h then stimulated with TSQ (TNF, Smac-mimetic, Q-VD-OPh) ( A ) or TSZ (TNF, Smac-mimetic, z-VAD-fmk) ( B ) for 24 h to induce necroptosis. Cell death was quantified by propidium iodide (PI) staining using flow cytometry. Data represent the mean of n = 3 ( A ) or n = 4 ( B ) independent experiments and error bars represent SEM. ( C – F ) Human U937 cells were treated with increasing concentrations of ABT-869 or control compounds, MLKL inhibitor NSA and RIPK1 inhibitor GSK′481, DMSO alone or left untreated (UT) for 1 h then stimulated with TSQ (TNF, Smac-mimetic, Q-VD-OPh) for 48 h ( C ) or TSI (TNF, Smac-mimetic, IDN-6556) for 24 h ( E ) to induce necroptosis. Parallel experiments were performed to assess protection of TSQ ( D ) or TSI ( F ) induced death in the presence of the ABT-869 analogue, WEHI-615. Cell death was monitored by SPY505 (live cells) and propidium iodide (PI; dead cells) uptake using IncuCyte live cell imaging. One representative result shown from n = 4 ( C , D ) or n = 3 ( E , F ) independent experiments. See also Supplementary Figure S2A–H.
    Figure Legend Snippet: ( A , B ) Wild-type mouse dermal fibroblast (MDF) cells were treated with increasing concentrations of ABT-869 or control compounds, RIPK3 inhibitors GSK′872 and GSK′843, DMSO alone or left untreated (UT) for 1 h then stimulated with TSQ (TNF, Smac-mimetic, Q-VD-OPh) ( A ) or TSZ (TNF, Smac-mimetic, z-VAD-fmk) ( B ) for 24 h to induce necroptosis. Cell death was quantified by propidium iodide (PI) staining using flow cytometry. Data represent the mean of n = 3 ( A ) or n = 4 ( B ) independent experiments and error bars represent SEM. ( C – F ) Human U937 cells were treated with increasing concentrations of ABT-869 or control compounds, MLKL inhibitor NSA and RIPK1 inhibitor GSK′481, DMSO alone or left untreated (UT) for 1 h then stimulated with TSQ (TNF, Smac-mimetic, Q-VD-OPh) for 48 h ( C ) or TSI (TNF, Smac-mimetic, IDN-6556) for 24 h ( E ) to induce necroptosis. Parallel experiments were performed to assess protection of TSQ ( D ) or TSI ( F ) induced death in the presence of the ABT-869 analogue, WEHI-615. Cell death was monitored by SPY505 (live cells) and propidium iodide (PI; dead cells) uptake using IncuCyte live cell imaging. One representative result shown from n = 4 ( C , D ) or n = 3 ( E , F ) independent experiments. See also Supplementary Figure S2A–H.

    Techniques Used: Staining, Flow Cytometry, Live Cell Imaging

    ( A ) Binding affinities ( K D ) of ABT-869 and WEHI-615 for human full-length MLKL, RIPK1 kinase domain and RIPK3 kinase domain measured by competition binding assays from the DiscoverX KINOME scan platform using the Kd ELECT service. Each value is the mean of two replicates. ( B – D ) Cellular Thermal Shift Assays (CETSA) in mouse and human cells. Mlkl −/− mouse dermal fibroblast (MDF) cells expressing MLKL Q343A ( B ), wild-type MDF cells ( C ) and human U937 cells ( D ) were treated with DMSO, ABT-869, WEHI-615, RIPK1 inhibitor Nec-1s, RIPK3 inhibitor GSK′872 or human RIPK1 inhibitor GSK′481 (all 20 µM). Cells were subjected to an increasing temperature gradient focused around the melting temperature of the protein of interest. Following the separation of soluble and insoluble proteins, the remaining soluble proteins were detected by Western blot. Red asterisks denote protein standards. One representative result shown from n = 3 ( B , C ) or n = 2–3 ( D ) independent experiments. See also Supplementary Figure S3A–C.
    Figure Legend Snippet: ( A ) Binding affinities ( K D ) of ABT-869 and WEHI-615 for human full-length MLKL, RIPK1 kinase domain and RIPK3 kinase domain measured by competition binding assays from the DiscoverX KINOME scan platform using the Kd ELECT service. Each value is the mean of two replicates. ( B – D ) Cellular Thermal Shift Assays (CETSA) in mouse and human cells. Mlkl −/− mouse dermal fibroblast (MDF) cells expressing MLKL Q343A ( B ), wild-type MDF cells ( C ) and human U937 cells ( D ) were treated with DMSO, ABT-869, WEHI-615, RIPK1 inhibitor Nec-1s, RIPK3 inhibitor GSK′872 or human RIPK1 inhibitor GSK′481 (all 20 µM). Cells were subjected to an increasing temperature gradient focused around the melting temperature of the protein of interest. Following the separation of soluble and insoluble proteins, the remaining soluble proteins were detected by Western blot. Red asterisks denote protein standards. One representative result shown from n = 3 ( B , C ) or n = 2–3 ( D ) independent experiments. See also Supplementary Figure S3A–C.

    Techniques Used: Binding Assay, Expressing, Western Blot

    Thermal Shift Assays (TSA) with mouse and human RIPK1 and RIPK3 kinase domains. Increasing concentrations of ABT-869 or WEHI-615 were tested for their ability to alter the melting temperature ( T M ) of mouse RIPK1 (9.5 µg) ( A , B ), human RIPK1 (12 µg) ( C , D ), mouse RIPK3 (10 µg) ( E , F ) and human RIPK3 (6.5 µg) ( G , H ) compared with the positive controls Compound 2 for mouse RIPK1, GSK′481 for human RIPK1 and GSK′872 for mouse and human RIPK3 (all 30 µM). Data represent the mean of n = 3 independent experiments and error bars represent SEM. See also Supplementary Figure S4A–H.
    Figure Legend Snippet: Thermal Shift Assays (TSA) with mouse and human RIPK1 and RIPK3 kinase domains. Increasing concentrations of ABT-869 or WEHI-615 were tested for their ability to alter the melting temperature ( T M ) of mouse RIPK1 (9.5 µg) ( A , B ), human RIPK1 (12 µg) ( C , D ), mouse RIPK3 (10 µg) ( E , F ) and human RIPK3 (6.5 µg) ( G , H ) compared with the positive controls Compound 2 for mouse RIPK1, GSK′481 for human RIPK1 and GSK′872 for mouse and human RIPK3 (all 30 µM). Data represent the mean of n = 3 independent experiments and error bars represent SEM. See also Supplementary Figure S4A–H.

    Techniques Used:

    ( A – H ) In vitro phosphorylation assays with mouse and human RIPK1 and RIPK3 kinase domains measured by ADP-Glo Kinase Assays. Increasing concentrations of ABT-869 or WEHI-615 were tested for their ability to inhibit the autophosphorylation (IC 50 ) of mouse RIPK1 (200 nM) ( A , B ), human RIPK1 (200 nM) ( C , D ), mouse RIPK3 (10 nM) ( E , F ) and human RIPK3 (10 nM) ( G , H ). Data represent the mean of n = 3 ( A , B , E , F ) or n = 2 ( C , D , G , H ) independent experiments and error bars represent SEM. ( I ) Cellular phosphorylation assays. Wild-type mouse dermal fibroblast (MDF) cells were treated with DMSO, ABT-869, WEHI-615, RIPK1 inhibitor Nec-1s or RIPK3 inhibitor GSK′872 for 2 h then stimulated with TSI (TNF, Smac-mimetic, IDN-6556) for 2 h to induce autophosphorylation of RIPK1 and RIPK3. Ripk1 −/− Mlkl −/− MDF cells and Ripk3 −/− MDF cells were included as controls. Phospho-RIPK1 and phospho-RIPK3 protein levels were detected from whole cell lysates by Western blot. Red asterisks denote protein standards. One representative result shown from n = 3 independent experiments. See also Supplementary Figure S5A–C.
    Figure Legend Snippet: ( A – H ) In vitro phosphorylation assays with mouse and human RIPK1 and RIPK3 kinase domains measured by ADP-Glo Kinase Assays. Increasing concentrations of ABT-869 or WEHI-615 were tested for their ability to inhibit the autophosphorylation (IC 50 ) of mouse RIPK1 (200 nM) ( A , B ), human RIPK1 (200 nM) ( C , D ), mouse RIPK3 (10 nM) ( E , F ) and human RIPK3 (10 nM) ( G , H ). Data represent the mean of n = 3 ( A , B , E , F ) or n = 2 ( C , D , G , H ) independent experiments and error bars represent SEM. ( I ) Cellular phosphorylation assays. Wild-type mouse dermal fibroblast (MDF) cells were treated with DMSO, ABT-869, WEHI-615, RIPK1 inhibitor Nec-1s or RIPK3 inhibitor GSK′872 for 2 h then stimulated with TSI (TNF, Smac-mimetic, IDN-6556) for 2 h to induce autophosphorylation of RIPK1 and RIPK3. Ripk1 −/− Mlkl −/− MDF cells and Ripk3 −/− MDF cells were included as controls. Phospho-RIPK1 and phospho-RIPK3 protein levels were detected from whole cell lysates by Western blot. Red asterisks denote protein standards. One representative result shown from n = 3 independent experiments. See also Supplementary Figure S5A–C.

    Techniques Used: In Vitro, Western Blot


    Figure Legend Snippet:

    Techniques Used: Western Blot, Produced, Transduction


    Figure Legend Snippet:

    Techniques Used:

    rabbit anti ripk3  (ProSci Incorporated)


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    ProSci Incorporated rabbit anti ripk3
    A , B U937 cell lines of various genotypes expressing an inducible MLKL(1-180)-gyrase fusion were treated with doxycycline (to induce expression) and/or coumermycin (to induce dimerisation), or left untreated. After 24 h ( A ) or 48 h ( B ), cells were harvested and assessed for cell death (PI uptake measured using flow cytometry), counting a minimum of 5000 cells. Data represent three independent experiments, with the exception of the BAX −/− BAK −/− cell lines, where n = 2. Statistics were calculated in GraphPad Prism 8, and p values are shown where <0.05. C The same U937 cells used in panel A and B were treated with doxycycline alone and left overnight. After approximately 16 h, cells were harvested and lysed for analysis of fusion protein expression via western blot. The upper MLKL band (*) represents endogenous MLKL, and the lower band (arrow) represents the MLKL (1-180) -gyrase fusion protein. D Wild-type U937 were treated with TNF (100 ng/ml), Smac-mimetic (0.5 µM), and caspase inhibitor IDN-6556 (emricasan; 5 µM) (TSI) to induce necroptosis or TNF and Smac-mimetic (TS) alone to induce apoptosis. In parallel, MLKL −/− U937 expressing an inducible MLKL (1-180) -gyrase fusion were treated with doxycycline and coumermycin in the presence or absence of necrosulfonamide (NSA). Images were taken at the specified timepoints using an IncuCyte S3 System to track morphological changes and PI uptake (shown by red). Cells with a necroptotic phenotype (cellular swelling) are indicate with yellow arrows. Three fields were examined per well with representative data shown. E Wild-type U937 cells were treated with doxycycline (20 nM) to induce expression of human wild-type <t>RIPK3</t> in the presence of various cell death inhibitors, as indicated, for 24 h. At the conclusion of the experiment, cells were analysed for PI uptake using flow cytometry, with a minimum of 5000 cells counted. Data represent four or more independent experiments. F Wild-type, MLKL −/− and RIPK1 −/− U937 expressing inducible human wild-type RIPK3 were treated with doxycycline (20 nM) and IDN-6556 (5 µM), doxycycline alone, or left untreated. After 24 h, cell death was measured as PI uptake using flow cytometry. Data are a summary of six independent experiments.
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    1) Product Images from "The Lck inhibitor, AMG-47a, blocks necroptosis and implicates RIPK1 in signalling downstream of MLKL"

    Article Title: The Lck inhibitor, AMG-47a, blocks necroptosis and implicates RIPK1 in signalling downstream of MLKL

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-022-04740-w

    A , B U937 cell lines of various genotypes expressing an inducible MLKL(1-180)-gyrase fusion were treated with doxycycline (to induce expression) and/or coumermycin (to induce dimerisation), or left untreated. After 24 h ( A ) or 48 h ( B ), cells were harvested and assessed for cell death (PI uptake measured using flow cytometry), counting a minimum of 5000 cells. Data represent three independent experiments, with the exception of the BAX −/− BAK −/− cell lines, where n = 2. Statistics were calculated in GraphPad Prism 8, and p values are shown where <0.05. C The same U937 cells used in panel A and B were treated with doxycycline alone and left overnight. After approximately 16 h, cells were harvested and lysed for analysis of fusion protein expression via western blot. The upper MLKL band (*) represents endogenous MLKL, and the lower band (arrow) represents the MLKL (1-180) -gyrase fusion protein. D Wild-type U937 were treated with TNF (100 ng/ml), Smac-mimetic (0.5 µM), and caspase inhibitor IDN-6556 (emricasan; 5 µM) (TSI) to induce necroptosis or TNF and Smac-mimetic (TS) alone to induce apoptosis. In parallel, MLKL −/− U937 expressing an inducible MLKL (1-180) -gyrase fusion were treated with doxycycline and coumermycin in the presence or absence of necrosulfonamide (NSA). Images were taken at the specified timepoints using an IncuCyte S3 System to track morphological changes and PI uptake (shown by red). Cells with a necroptotic phenotype (cellular swelling) are indicate with yellow arrows. Three fields were examined per well with representative data shown. E Wild-type U937 cells were treated with doxycycline (20 nM) to induce expression of human wild-type RIPK3 in the presence of various cell death inhibitors, as indicated, for 24 h. At the conclusion of the experiment, cells were analysed for PI uptake using flow cytometry, with a minimum of 5000 cells counted. Data represent four or more independent experiments. F Wild-type, MLKL −/− and RIPK1 −/− U937 expressing inducible human wild-type RIPK3 were treated with doxycycline (20 nM) and IDN-6556 (5 µM), doxycycline alone, or left untreated. After 24 h, cell death was measured as PI uptake using flow cytometry. Data are a summary of six independent experiments.
    Figure Legend Snippet: A , B U937 cell lines of various genotypes expressing an inducible MLKL(1-180)-gyrase fusion were treated with doxycycline (to induce expression) and/or coumermycin (to induce dimerisation), or left untreated. After 24 h ( A ) or 48 h ( B ), cells were harvested and assessed for cell death (PI uptake measured using flow cytometry), counting a minimum of 5000 cells. Data represent three independent experiments, with the exception of the BAX −/− BAK −/− cell lines, where n = 2. Statistics were calculated in GraphPad Prism 8, and p values are shown where <0.05. C The same U937 cells used in panel A and B were treated with doxycycline alone and left overnight. After approximately 16 h, cells were harvested and lysed for analysis of fusion protein expression via western blot. The upper MLKL band (*) represents endogenous MLKL, and the lower band (arrow) represents the MLKL (1-180) -gyrase fusion protein. D Wild-type U937 were treated with TNF (100 ng/ml), Smac-mimetic (0.5 µM), and caspase inhibitor IDN-6556 (emricasan; 5 µM) (TSI) to induce necroptosis or TNF and Smac-mimetic (TS) alone to induce apoptosis. In parallel, MLKL −/− U937 expressing an inducible MLKL (1-180) -gyrase fusion were treated with doxycycline and coumermycin in the presence or absence of necrosulfonamide (NSA). Images were taken at the specified timepoints using an IncuCyte S3 System to track morphological changes and PI uptake (shown by red). Cells with a necroptotic phenotype (cellular swelling) are indicate with yellow arrows. Three fields were examined per well with representative data shown. E Wild-type U937 cells were treated with doxycycline (20 nM) to induce expression of human wild-type RIPK3 in the presence of various cell death inhibitors, as indicated, for 24 h. At the conclusion of the experiment, cells were analysed for PI uptake using flow cytometry, with a minimum of 5000 cells counted. Data represent four or more independent experiments. F Wild-type, MLKL −/− and RIPK1 −/− U937 expressing inducible human wild-type RIPK3 were treated with doxycycline (20 nM) and IDN-6556 (5 µM), doxycycline alone, or left untreated. After 24 h, cell death was measured as PI uptake using flow cytometry. Data are a summary of six independent experiments.

    Techniques Used: Expressing, Flow Cytometry, Western Blot

    A Three different experimental protocols were used to assess the ability of AMG-47a to inhibit cell death caused by the expression and dimerisation of the MLKL (1-180) -gyrase fusion protein. In protocol 1, inhibitors (or DMSO) were added first, followed by doxycycline and coumermycin together, so that the fusion protein could dimerise on expression. In protocol 2, addition of coumermycin was delayed to allow levels of the fusion protein to accumulate before dimerisation was induced. In protocol 3, the addition of inhibitors (or DMSO) was also delayed, so that it was added after the fusion protein had been expressed for 16 h, but before the addition of coumermycin. In all experimental methods, cells were harvested 24 h after the experiment was initiated. B Wild-type U937 cells expressing the MLKL (1-180) -gyrase fusion protein were treated using either protocol 1, 2, or 3, as described above ( A ). At the conclusion of the experiment, cells were analysed for PI uptake using flow cytometry, with a minimum of 5000 cells counted. Data represent three independent experiments; bars indicate the mean and error bars indicate standard error of the mean. Statistics were calculated in GraphPad Prism8, and p values are shown where <0.05. C Wild-type U937 cells with inducible human RIPK3 were treated with increasing doses of AMG-47a in the presence of 40 nM doxycycline, to induce expression, and 5 µM IDN-6556, to block caspase-dependent cell death. After 48 h, cells were harvested and viability assessed using the Cell-Titer Glo 2 (Promega) system. Data represent two independent experiments (four replicates per experiment) and have been normalised against doxycycline plus IDN-6556 (0% viability) and doxycycline plus IDN-6556 plus NSA (100% viability).
    Figure Legend Snippet: A Three different experimental protocols were used to assess the ability of AMG-47a to inhibit cell death caused by the expression and dimerisation of the MLKL (1-180) -gyrase fusion protein. In protocol 1, inhibitors (or DMSO) were added first, followed by doxycycline and coumermycin together, so that the fusion protein could dimerise on expression. In protocol 2, addition of coumermycin was delayed to allow levels of the fusion protein to accumulate before dimerisation was induced. In protocol 3, the addition of inhibitors (or DMSO) was also delayed, so that it was added after the fusion protein had been expressed for 16 h, but before the addition of coumermycin. In all experimental methods, cells were harvested 24 h after the experiment was initiated. B Wild-type U937 cells expressing the MLKL (1-180) -gyrase fusion protein were treated using either protocol 1, 2, or 3, as described above ( A ). At the conclusion of the experiment, cells were analysed for PI uptake using flow cytometry, with a minimum of 5000 cells counted. Data represent three independent experiments; bars indicate the mean and error bars indicate standard error of the mean. Statistics were calculated in GraphPad Prism8, and p values are shown where <0.05. C Wild-type U937 cells with inducible human RIPK3 were treated with increasing doses of AMG-47a in the presence of 40 nM doxycycline, to induce expression, and 5 µM IDN-6556, to block caspase-dependent cell death. After 48 h, cells were harvested and viability assessed using the Cell-Titer Glo 2 (Promega) system. Data represent two independent experiments (four replicates per experiment) and have been normalised against doxycycline plus IDN-6556 (0% viability) and doxycycline plus IDN-6556 plus NSA (100% viability).

    Techniques Used: Expressing, Flow Cytometry, Blocking Assay

    A, B, C U937, HT29 and MDF cell lines were treated with DMSO, AMG-47a (1 µM for U937 and MDF; 2 µM for HT29), TSI, or a combination of AMG-47a and TSI over a time course as indicated in the individual figure panels. At the conclusion of the experiment, cells were harvested and lysed for western blot analysis. All cell lines were assessed for phosphorylation and total protein of the three key necroptosis effector proteins, RIPK1, RIPK3, and MLKL, and total β-actin was used as a loading control. Where multiple bands are present, the specific band of interest is indicated with a red arrow. Data are representative of at least three independent experiments. U937 ( D ) or MDF ( E ) cell lines were treated with AMG-47a at 10 µM, the RIPK1 inhibitors (GSK-481, Nec-1s) or a RIPK3 inhibitor (GSK-872) at 20 µM, or an equivalent amount of DMSO, for 1 h at 37 °C. Cell suspensions were then heated over an increasing temperature gradient (as indicated in the methods) for 3 min. Cells were then lysed, and the amount of remaining soluble protein at each temperature point was analysed using Western blot. Results are representative of two (RIPK1 and RIPK3 in U937) or three (all other analyses) independent experiments. AMG-47a binding to RIPK1 ( F ) and RIPK3 ( G ) was measured in competitive binding assays using the KINOMEscan ® Assay Platform (DiscoverX). Graphs were plotted using the raw data supplied from DiscoverX, and a curve was fitted using non-linear regression (GraphPad Prism) to determine the K d and associated error (95% confidence interval). Data represent one (RIPK3) or two (RIPK1) independent experiments, with two runs performed in all experiments and each colour on the graph representing a separate run. All data points were used for the non-linear regression. K d determinations for individual replicates can be found in supplementary data (Supplementary Fig. ). H , I AMG-47a was serially diluted from a maximum concentration of 50 µM (RIPK1) or 100 µM (RIPK3), and its ability to inhibit human RIPK1 or RIPK3 kinase activity was assessed using the ADP-Glo Kinase Assay (Promega). The reaction was allowed to proceed for 4 h before readouts were performed, and data were normalised to DMSO (0% inhibition) and either 1 µM GSK-481 (100% inhibition of RIPK1) or 1 µM GSK-872 (100% inhibition of RIPK3). Data are a summary of two independent experiments performed in duplicate, and the IC 50 and associated error (95% confidence interval) were determined using non-linear regression, as above. Each colour on the graph represents one independent experiment.
    Figure Legend Snippet: A, B, C U937, HT29 and MDF cell lines were treated with DMSO, AMG-47a (1 µM for U937 and MDF; 2 µM for HT29), TSI, or a combination of AMG-47a and TSI over a time course as indicated in the individual figure panels. At the conclusion of the experiment, cells were harvested and lysed for western blot analysis. All cell lines were assessed for phosphorylation and total protein of the three key necroptosis effector proteins, RIPK1, RIPK3, and MLKL, and total β-actin was used as a loading control. Where multiple bands are present, the specific band of interest is indicated with a red arrow. Data are representative of at least three independent experiments. U937 ( D ) or MDF ( E ) cell lines were treated with AMG-47a at 10 µM, the RIPK1 inhibitors (GSK-481, Nec-1s) or a RIPK3 inhibitor (GSK-872) at 20 µM, or an equivalent amount of DMSO, for 1 h at 37 °C. Cell suspensions were then heated over an increasing temperature gradient (as indicated in the methods) for 3 min. Cells were then lysed, and the amount of remaining soluble protein at each temperature point was analysed using Western blot. Results are representative of two (RIPK1 and RIPK3 in U937) or three (all other analyses) independent experiments. AMG-47a binding to RIPK1 ( F ) and RIPK3 ( G ) was measured in competitive binding assays using the KINOMEscan ® Assay Platform (DiscoverX). Graphs were plotted using the raw data supplied from DiscoverX, and a curve was fitted using non-linear regression (GraphPad Prism) to determine the K d and associated error (95% confidence interval). Data represent one (RIPK3) or two (RIPK1) independent experiments, with two runs performed in all experiments and each colour on the graph representing a separate run. All data points were used for the non-linear regression. K d determinations for individual replicates can be found in supplementary data (Supplementary Fig. ). H , I AMG-47a was serially diluted from a maximum concentration of 50 µM (RIPK1) or 100 µM (RIPK3), and its ability to inhibit human RIPK1 or RIPK3 kinase activity was assessed using the ADP-Glo Kinase Assay (Promega). The reaction was allowed to proceed for 4 h before readouts were performed, and data were normalised to DMSO (0% inhibition) and either 1 µM GSK-481 (100% inhibition of RIPK1) or 1 µM GSK-872 (100% inhibition of RIPK3). Data are a summary of two independent experiments performed in duplicate, and the IC 50 and associated error (95% confidence interval) were determined using non-linear regression, as above. Each colour on the graph represents one independent experiment.

    Techniques Used: Western Blot, Binding Assay, Concentration Assay, Activity Assay, Kinase Assay, Inhibition

    A The cell death induced by forced dimerisation of the human MLKL NTD involves two distinct processes. In the first, MLKL can self-associate into discrete oligomers on addition of doxycycline. In the second, addition of coumermycin promotes the assembly of large multimeric complexes. AMG-47a is unable to inhibit the second process, thus we propose it prevents membrane attachment, translocation, or oligomerisation of the human MLKL NTD through its activity as a kinase inhibitor. As AMG-47a binds RIPK1, and as RIPK1 is required for this form of cell death, we propose the primary target of AMG-47a is RIPK1 in this context. B When a cell receives a necroptotic stimulus, a number of key events occur, beginning with assembly of a necrosome containing RIPK1, RIPK3, and MLKL. This triggers a number of events, beginning with the phosphorylation of MLKL by RIPK3, which enables a conformational change in MLKL that allows oligomerisation, translocation to cellular membranes, and ultimately cell death. Our data suggest an additional role for RIPK1 in human necroptosis by supporting the conformational change, oligomerisation, or membrane translocation of MLKL following phosphorylation by RIPK3.
    Figure Legend Snippet: A The cell death induced by forced dimerisation of the human MLKL NTD involves two distinct processes. In the first, MLKL can self-associate into discrete oligomers on addition of doxycycline. In the second, addition of coumermycin promotes the assembly of large multimeric complexes. AMG-47a is unable to inhibit the second process, thus we propose it prevents membrane attachment, translocation, or oligomerisation of the human MLKL NTD through its activity as a kinase inhibitor. As AMG-47a binds RIPK1, and as RIPK1 is required for this form of cell death, we propose the primary target of AMG-47a is RIPK1 in this context. B When a cell receives a necroptotic stimulus, a number of key events occur, beginning with assembly of a necrosome containing RIPK1, RIPK3, and MLKL. This triggers a number of events, beginning with the phosphorylation of MLKL by RIPK3, which enables a conformational change in MLKL that allows oligomerisation, translocation to cellular membranes, and ultimately cell death. Our data suggest an additional role for RIPK1 in human necroptosis by supporting the conformational change, oligomerisation, or membrane translocation of MLKL following phosphorylation by RIPK3.

    Techniques Used: Translocation Assay, Activity Assay

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    ProSci Incorporated rabbit anti ripk3
    RAGE mediates DAMP release following transfusion and RAGE deletion attenuates necroptotic cell death. (a) Plasma HMGB1 and <t>RIPK3</t> following RBC transfusion in WT and RAGE KO mice (n = 3 from 2 independent experiments and * P <0.002, **P = 0.023 for HMGB1; n = 3 from three independent experiments and *P = 0.003, **P = 0.02 for RIPK3). (b) HUVEC were treated with TNFα (100 ng/ml), LPS (10 ng/ml), or CpG (10 μg/ml) in the presence of zVAD for 4 h. RAGE knockdown led to a significant attenuation in cell death following TNFα-zVAD, LPS-zVAD and CpG-zVAD treatment (n = 4 from two independent experiments, P = 0.036 for TNFα, P = 0.035 for LPS, P = 0.005 for CpG). (c) RAGE and RIPK3 expression in naive or TNFα-ZVAD treated HMVEC-L. Proximity Ligation Assay of RAGE and RIPK3 in naive and TNFα-zVAD treated HMVEC-L, RAGE-RIPK3-RED, F-Actin-Green, DAPI-Blue.
    Rabbit Anti Ripk3, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "RAGE interacts with the necroptotic protein RIPK3 and mediates transfusion-induced danger signal release"

    Article Title: RAGE interacts with the necroptotic protein RIPK3 and mediates transfusion-induced danger signal release

    Journal: Vox sanguinis

    doi: 10.1111/vox.12946

    RAGE mediates DAMP release following transfusion and RAGE deletion attenuates necroptotic cell death. (a) Plasma HMGB1 and RIPK3 following RBC transfusion in WT and RAGE KO mice (n = 3 from 2 independent experiments and * P <0.002, **P = 0.023 for HMGB1; n = 3 from three independent experiments and *P = 0.003, **P = 0.02 for RIPK3). (b) HUVEC were treated with TNFα (100 ng/ml), LPS (10 ng/ml), or CpG (10 μg/ml) in the presence of zVAD for 4 h. RAGE knockdown led to a significant attenuation in cell death following TNFα-zVAD, LPS-zVAD and CpG-zVAD treatment (n = 4 from two independent experiments, P = 0.036 for TNFα, P = 0.035 for LPS, P = 0.005 for CpG). (c) RAGE and RIPK3 expression in naive or TNFα-ZVAD treated HMVEC-L. Proximity Ligation Assay of RAGE and RIPK3 in naive and TNFα-zVAD treated HMVEC-L, RAGE-RIPK3-RED, F-Actin-Green, DAPI-Blue.
    Figure Legend Snippet: RAGE mediates DAMP release following transfusion and RAGE deletion attenuates necroptotic cell death. (a) Plasma HMGB1 and RIPK3 following RBC transfusion in WT and RAGE KO mice (n = 3 from 2 independent experiments and * P <0.002, **P = 0.023 for HMGB1; n = 3 from three independent experiments and *P = 0.003, **P = 0.02 for RIPK3). (b) HUVEC were treated with TNFα (100 ng/ml), LPS (10 ng/ml), or CpG (10 μg/ml) in the presence of zVAD for 4 h. RAGE knockdown led to a significant attenuation in cell death following TNFα-zVAD, LPS-zVAD and CpG-zVAD treatment (n = 4 from two independent experiments, P = 0.036 for TNFα, P = 0.035 for LPS, P = 0.005 for CpG). (c) RAGE and RIPK3 expression in naive or TNFα-ZVAD treated HMVEC-L. Proximity Ligation Assay of RAGE and RIPK3 in naive and TNFα-zVAD treated HMVEC-L, RAGE-RIPK3-RED, F-Actin-Green, DAPI-Blue.

    Techniques Used: Expressing, Proximity Ligation Assay

    RAGE and RIPK3 are detected in the nucleus of HMVEC-L. (a) HMVEC-L were transfected with GFP-tagged RAGE. Anti-GFP beads were utilized to pulldown RAGE from membrane (M), nuclear (N) or cytosolic fractions (c). Immunoblotting for RAGE and RIPK3 was performed. (b) HMVEC-L were treated with TNFα-zVAD or RBC-zVAD for 4 h. Cell lysates were fractionated and probed for RIPK3 or RAGE. RAGE and RIPK3 were detected in all three compartments. (c) RAGE and RIPK3 expression in naive, RBC-zVAD, or TNFα-ZVAD treated HMVEC-L (n = 6 from two independent experiments, representative micrographs shown).
    Figure Legend Snippet: RAGE and RIPK3 are detected in the nucleus of HMVEC-L. (a) HMVEC-L were transfected with GFP-tagged RAGE. Anti-GFP beads were utilized to pulldown RAGE from membrane (M), nuclear (N) or cytosolic fractions (c). Immunoblotting for RAGE and RIPK3 was performed. (b) HMVEC-L were treated with TNFα-zVAD or RBC-zVAD for 4 h. Cell lysates were fractionated and probed for RIPK3 or RAGE. RAGE and RIPK3 were detected in all three compartments. (c) RAGE and RIPK3 expression in naive, RBC-zVAD, or TNFα-ZVAD treated HMVEC-L (n = 6 from two independent experiments, representative micrographs shown).

    Techniques Used: Transfection, Western Blot, Expressing

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    ProSci Incorporated rabbit anti ripk3
    RAGE mediates DAMP release following transfusion and RAGE deletion attenuates necroptotic cell death. (a) Plasma HMGB1 and <t>RIPK3</t> following RBC transfusion in WT and RAGE KO mice (n = 3 from 2 independent experiments and * P <0.002, **P = 0.023 for HMGB1; n = 3 from three independent experiments and *P = 0.003, **P = 0.02 for RIPK3). (b) HUVEC were treated with TNFα (100 ng/ml), LPS (10 ng/ml), or CpG (10 μg/ml) in the presence of zVAD for 4 h. RAGE knockdown led to a significant attenuation in cell death following TNFα-zVAD, LPS-zVAD and CpG-zVAD treatment (n = 4 from two independent experiments, P = 0.036 for TNFα, P = 0.035 for LPS, P = 0.005 for CpG). (c) RAGE and RIPK3 expression in naive or TNFα-ZVAD treated HMVEC-L. Proximity Ligation Assay of RAGE and RIPK3 in naive and TNFα-zVAD treated HMVEC-L, RAGE-RIPK3-RED, F-Actin-Green, DAPI-Blue.
    Rabbit Anti Ripk3, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "RAGE interacts with the necroptotic protein RIPK3 and mediates transfusion-induced danger signal release"

    Article Title: RAGE interacts with the necroptotic protein RIPK3 and mediates transfusion-induced danger signal release

    Journal: Vox sanguinis

    doi: 10.1111/vox.12946

    RAGE mediates DAMP release following transfusion and RAGE deletion attenuates necroptotic cell death. (a) Plasma HMGB1 and RIPK3 following RBC transfusion in WT and RAGE KO mice (n = 3 from 2 independent experiments and * P <0.002, **P = 0.023 for HMGB1; n = 3 from three independent experiments and *P = 0.003, **P = 0.02 for RIPK3). (b) HUVEC were treated with TNFα (100 ng/ml), LPS (10 ng/ml), or CpG (10 μg/ml) in the presence of zVAD for 4 h. RAGE knockdown led to a significant attenuation in cell death following TNFα-zVAD, LPS-zVAD and CpG-zVAD treatment (n = 4 from two independent experiments, P = 0.036 for TNFα, P = 0.035 for LPS, P = 0.005 for CpG). (c) RAGE and RIPK3 expression in naive or TNFα-ZVAD treated HMVEC-L. Proximity Ligation Assay of RAGE and RIPK3 in naive and TNFα-zVAD treated HMVEC-L, RAGE-RIPK3-RED, F-Actin-Green, DAPI-Blue.
    Figure Legend Snippet: RAGE mediates DAMP release following transfusion and RAGE deletion attenuates necroptotic cell death. (a) Plasma HMGB1 and RIPK3 following RBC transfusion in WT and RAGE KO mice (n = 3 from 2 independent experiments and * P <0.002, **P = 0.023 for HMGB1; n = 3 from three independent experiments and *P = 0.003, **P = 0.02 for RIPK3). (b) HUVEC were treated with TNFα (100 ng/ml), LPS (10 ng/ml), or CpG (10 μg/ml) in the presence of zVAD for 4 h. RAGE knockdown led to a significant attenuation in cell death following TNFα-zVAD, LPS-zVAD and CpG-zVAD treatment (n = 4 from two independent experiments, P = 0.036 for TNFα, P = 0.035 for LPS, P = 0.005 for CpG). (c) RAGE and RIPK3 expression in naive or TNFα-ZVAD treated HMVEC-L. Proximity Ligation Assay of RAGE and RIPK3 in naive and TNFα-zVAD treated HMVEC-L, RAGE-RIPK3-RED, F-Actin-Green, DAPI-Blue.

    Techniques Used: Expressing, Proximity Ligation Assay

    RAGE and RIPK3 are detected in the nucleus of HMVEC-L. (a) HMVEC-L were transfected with GFP-tagged RAGE. Anti-GFP beads were utilized to pulldown RAGE from membrane (M), nuclear (N) or cytosolic fractions (c). Immunoblotting for RAGE and RIPK3 was performed. (b) HMVEC-L were treated with TNFα-zVAD or RBC-zVAD for 4 h. Cell lysates were fractionated and probed for RIPK3 or RAGE. RAGE and RIPK3 were detected in all three compartments. (c) RAGE and RIPK3 expression in naive, RBC-zVAD, or TNFα-ZVAD treated HMVEC-L (n = 6 from two independent experiments, representative micrographs shown).
    Figure Legend Snippet: RAGE and RIPK3 are detected in the nucleus of HMVEC-L. (a) HMVEC-L were transfected with GFP-tagged RAGE. Anti-GFP beads were utilized to pulldown RAGE from membrane (M), nuclear (N) or cytosolic fractions (c). Immunoblotting for RAGE and RIPK3 was performed. (b) HMVEC-L were treated with TNFα-zVAD or RBC-zVAD for 4 h. Cell lysates were fractionated and probed for RIPK3 or RAGE. RAGE and RIPK3 were detected in all three compartments. (c) RAGE and RIPK3 expression in naive, RBC-zVAD, or TNFα-ZVAD treated HMVEC-L (n = 6 from two independent experiments, representative micrographs shown).

    Techniques Used: Transfection, Western Blot, Expressing

    rabbit polyclonal anti ripk3  (ProSci Incorporated)


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    ProSci Incorporated rabbit polyclonal anti ripk3
    (A) IAV-induced cell death kinetics in primary wild-type (WT), Zbp1−/−, and <t>Ripk3−/−</t> MEFs.
    Rabbit Polyclonal Anti Ripk3, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Influenza Virus Z-RNAs Induce ZBP1-Mediated Necroptosis"

    Article Title: Influenza Virus Z-RNAs Induce ZBP1-Mediated Necroptosis

    Journal: Cell

    doi: 10.1016/j.cell.2020.02.050

    (A) IAV-induced cell death kinetics in primary wild-type (WT), Zbp1−/−, and Ripk3−/− MEFs.
    Figure Legend Snippet: (A) IAV-induced cell death kinetics in primary wild-type (WT), Zbp1−/−, and Ripk3−/− MEFs.

    Techniques Used:

    (A) IAV-infected (PR8, MOI = 2) WT MEFs were lysed at the indicated times p.i., separated into nuclear and cytoplasmic fractions, and examined for ZBP1, MLKL, RIPK3, and viral proteins by immunoblotting. Immunoblotting for GAPDH and histone H3 was used to confirm purity of cytoplasmic and nuclear fractions.
    Figure Legend Snippet: (A) IAV-infected (PR8, MOI = 2) WT MEFs were lysed at the indicated times p.i., separated into nuclear and cytoplasmic fractions, and examined for ZBP1, MLKL, RIPK3, and viral proteins by immunoblotting. Immunoblotting for GAPDH and histone H3 was used to confirm purity of cytoplasmic and nuclear fractions.

    Techniques Used: Infection, Western Blot

    (A) Survival analysis of age- and sex-matched WT (C57BL/6J) (n = 9), Zbp1−/− (n = 9), Ripk3−/− (n = 9), and Mlkl−/− (n = 8) mice infected with a modestly lethal (~LD20) dose of IAV (PR8, 2,500 EID50/mouse intranasally [i.n.]).
    Figure Legend Snippet: (A) Survival analysis of age- and sex-matched WT (C57BL/6J) (n = 9), Zbp1−/− (n = 9), Ripk3−/− (n = 9), and Mlkl−/− (n = 8) mice infected with a modestly lethal (~LD20) dose of IAV (PR8, 2,500 EID50/mouse intranasally [i.n.]).

    Techniques Used: Infection

    KEY RESOURCES TABLE
    Figure Legend Snippet: KEY RESOURCES TABLE

    Techniques Used: Recombinant, Enzyme-linked Immunosorbent Assay, SYBR Green Assay, Immunoprecipitation, RNA Sequencing Assay, Plasmid Preparation, Software

    rabbit anti ripk3  (ProSci Incorporated)


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    ProSci Incorporated rabbit anti ripk3
    a Human <t>RIPK3</t> domain architecture showing the immunogens or epitopes for the tested anti-RIPK3 antibodies. b Quantitation of the percentage of gated signals for the tested RIPK3 antibodies. c Quantitation of specific signal abundance produced by the tested RIPK3 antibodies on methanol-fixed (MeOH) or paraformaldehyde-fixed (PFA) HT29 cells. The number of cells imaged (N) to generate each signal-to-noise curve is shown. d Micrographs of immunofluorescent signals for the tested RIPK3 antibodies on HT29 cells. As indicated by each pseudocolour look-up-table, only immunosignals within the respective gate in panel c were visualised. Data are representative of n=3 (Cell Signaling Technology clone D6W2T and in-house clone 1H2) and n=2 (ProSci 2283, Novus Biological NBP2-24588 and MAB7604/clone 780115) independent experiments. Nuclei were detected by Hoechst 33342 staining and are demarked by white outlines in micrographs. e Immunoblot using the tested RIPK3 antibodies against wild-type and RIPK3 -/- HT29 cell lysates. Closed arrowheads indicate the main specific band. Open arrowheads indicate other specific bands of interest. Immunoblots were re-probed for GAPDH as loading control.
    Rabbit Anti Ripk3, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "A toolbox for imaging RIPK1, RIPK3 and MLKL in mouse and human cells"

    Article Title: A toolbox for imaging RIPK1, RIPK3 and MLKL in mouse and human cells

    Journal: bioRxiv

    doi: 10.1101/2020.10.26.356063

    a Human RIPK3 domain architecture showing the immunogens or epitopes for the tested anti-RIPK3 antibodies. b Quantitation of the percentage of gated signals for the tested RIPK3 antibodies. c Quantitation of specific signal abundance produced by the tested RIPK3 antibodies on methanol-fixed (MeOH) or paraformaldehyde-fixed (PFA) HT29 cells. The number of cells imaged (N) to generate each signal-to-noise curve is shown. d Micrographs of immunofluorescent signals for the tested RIPK3 antibodies on HT29 cells. As indicated by each pseudocolour look-up-table, only immunosignals within the respective gate in panel c were visualised. Data are representative of n=3 (Cell Signaling Technology clone D6W2T and in-house clone 1H2) and n=2 (ProSci 2283, Novus Biological NBP2-24588 and MAB7604/clone 780115) independent experiments. Nuclei were detected by Hoechst 33342 staining and are demarked by white outlines in micrographs. e Immunoblot using the tested RIPK3 antibodies against wild-type and RIPK3 -/- HT29 cell lysates. Closed arrowheads indicate the main specific band. Open arrowheads indicate other specific bands of interest. Immunoblots were re-probed for GAPDH as loading control.
    Figure Legend Snippet: a Human RIPK3 domain architecture showing the immunogens or epitopes for the tested anti-RIPK3 antibodies. b Quantitation of the percentage of gated signals for the tested RIPK3 antibodies. c Quantitation of specific signal abundance produced by the tested RIPK3 antibodies on methanol-fixed (MeOH) or paraformaldehyde-fixed (PFA) HT29 cells. The number of cells imaged (N) to generate each signal-to-noise curve is shown. d Micrographs of immunofluorescent signals for the tested RIPK3 antibodies on HT29 cells. As indicated by each pseudocolour look-up-table, only immunosignals within the respective gate in panel c were visualised. Data are representative of n=3 (Cell Signaling Technology clone D6W2T and in-house clone 1H2) and n=2 (ProSci 2283, Novus Biological NBP2-24588 and MAB7604/clone 780115) independent experiments. Nuclei were detected by Hoechst 33342 staining and are demarked by white outlines in micrographs. e Immunoblot using the tested RIPK3 antibodies against wild-type and RIPK3 -/- HT29 cell lysates. Closed arrowheads indicate the main specific band. Open arrowheads indicate other specific bands of interest. Immunoblots were re-probed for GAPDH as loading control.

    Techniques Used: Quantitation Assay, Produced, Staining, Western Blot

    a Mouse RIPK3 domain architecture showing the immunogens or epitopes for the tested anti-RIPK3 antibodies. b Quantitation of the percentage of gated signals for the tested RIPK3 antibodies. c Quantitation of specific signal abundance produced by the tested RIPK3 antibodies on methanol-fixed (MeOH) or paraformaldehyde-fixed (PFA) MDFs. The number of cells imaged (N) to generate each signal-to-noise curve is shown. d Micrographs of immunofluorescent signals for the tested RIPK3 antibodies on MDFs. As indicated by each pseudocolour look-up-table, only immunosignals within the respective gate in panel c were visualised. Data are representative of n=3 (in-house clone 8G7 and Genentech clone GEN135-35-9) and n=2 (ProSci 2283, in-house clone 1H12) independent experiments. Nuclei were detected by Hoechst 33342 staining and are demarked by white outlines in micrographs. e Immunoblot using the tested RIPK3 antibodies against lysates from wild-type and Ripk3 -/- MDFs. Closed arrowheads indicate the main specific band. Open arrowheads indicate other specific bands of interest. Immunoblots were re-probed for GAPDH as loading control.
    Figure Legend Snippet: a Mouse RIPK3 domain architecture showing the immunogens or epitopes for the tested anti-RIPK3 antibodies. b Quantitation of the percentage of gated signals for the tested RIPK3 antibodies. c Quantitation of specific signal abundance produced by the tested RIPK3 antibodies on methanol-fixed (MeOH) or paraformaldehyde-fixed (PFA) MDFs. The number of cells imaged (N) to generate each signal-to-noise curve is shown. d Micrographs of immunofluorescent signals for the tested RIPK3 antibodies on MDFs. As indicated by each pseudocolour look-up-table, only immunosignals within the respective gate in panel c were visualised. Data are representative of n=3 (in-house clone 8G7 and Genentech clone GEN135-35-9) and n=2 (ProSci 2283, in-house clone 1H12) independent experiments. Nuclei were detected by Hoechst 33342 staining and are demarked by white outlines in micrographs. e Immunoblot using the tested RIPK3 antibodies against lysates from wild-type and Ripk3 -/- MDFs. Closed arrowheads indicate the main specific band. Open arrowheads indicate other specific bands of interest. Immunoblots were re-probed for GAPDH as loading control.

    Techniques Used: Quantitation Assay, Produced, Staining, Western Blot

    rabbit anti ripk3  (ProSci Incorporated)


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    ProSci Incorporated rabbit anti ripk3
    Combined Loss of Caspases-1, -11, -12, and -8 (plus <t>RIPK3)</t> Leads to Lack of Bacterial Control upon Salmonella Infection (A) Bacterial replication over time in WT and Casp1 –/– ;Casp11 –/– mice infected with Salmonella Δ AroA (200 CFU). n = 10−22 mice per group per time point. Mean and SEM are shown. ∗∗ p < 0.005, ∗ p < 0.05, ns p > 0.05 = not significant. (B) Bacterial loads in spleen and liver of mice of the indicated genotypes 3 weeks post-infection with Salmonella Δ AroA (200 CFU). n = 7−48 mice per genotype. Mean and SEM are shown. ∗∗ p < 0.005, ∗ p < 0.05, ns p > 0.05 = not significant. (C) Bacterial loads in spleen and liver from mice of the indicated genotypes 1 to 3 weeks post-infection with Salmonella Δ AroA (200 CFU). n = 3−4 mice per genotype and time point. Mean and SEM are shown. ∗∗ p < 0.005, ∗ p < 0.05, ns p > 0.05 = not significant. (D) Mouse survival curves and corresponding bacterial loads in the spleen and liver at time of sacrifice in WT and Casp1 –/– ;Casp11 –/– ;Casp12 –/– ;Casp8 –/– ;Ripk3 −/− mice infected with Salmonella Δ AroA (200 CFU). n = 7−8 mice per genotype. Mean and SEM are shown. ∗∗ p < 0.005. (E) Bone marrow chimeras of the indicated genotypes were infected with Salmonella Δ AroA (200 CFU) and culled for analysis of bacterial loads in spleen and liver 3 weeks post-infection. n = 10 mice per group. Mean and SEM are shown. ∗∗ p < 0.005. Please also see <xref ref-type=Figure S1 . " width="250" height="auto" />
    Rabbit Anti Ripk3, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Flexible Usage and Interconnectivity of Diverse Cell Death Pathways Protect against Intracellular Infection"

    Article Title: Flexible Usage and Interconnectivity of Diverse Cell Death Pathways Protect against Intracellular Infection

    Journal: Immunity

    doi: 10.1016/j.immuni.2020.07.004

    Combined Loss of Caspases-1, -11, -12, and -8 (plus RIPK3) Leads to Lack of Bacterial Control upon Salmonella Infection (A) Bacterial replication over time in WT and Casp1 –/– ;Casp11 –/– mice infected with Salmonella Δ AroA (200 CFU). n = 10−22 mice per group per time point. Mean and SEM are shown. ∗∗ p < 0.005, ∗ p < 0.05, ns p > 0.05 = not significant. (B) Bacterial loads in spleen and liver of mice of the indicated genotypes 3 weeks post-infection with Salmonella Δ AroA (200 CFU). n = 7−48 mice per genotype. Mean and SEM are shown. ∗∗ p < 0.005, ∗ p < 0.05, ns p > 0.05 = not significant. (C) Bacterial loads in spleen and liver from mice of the indicated genotypes 1 to 3 weeks post-infection with Salmonella Δ AroA (200 CFU). n = 3−4 mice per genotype and time point. Mean and SEM are shown. ∗∗ p < 0.005, ∗ p < 0.05, ns p > 0.05 = not significant. (D) Mouse survival curves and corresponding bacterial loads in the spleen and liver at time of sacrifice in WT and Casp1 –/– ;Casp11 –/– ;Casp12 –/– ;Casp8 –/– ;Ripk3 −/− mice infected with Salmonella Δ AroA (200 CFU). n = 7−8 mice per genotype. Mean and SEM are shown. ∗∗ p < 0.005. (E) Bone marrow chimeras of the indicated genotypes were infected with Salmonella Δ AroA (200 CFU) and culled for analysis of bacterial loads in spleen and liver 3 weeks post-infection. n = 10 mice per group. Mean and SEM are shown. ∗∗ p < 0.005. Please also see <xref ref-type=Figure S1 . " title="... Loss of Caspases-1, -11, -12, and -8 (plus RIPK3) Leads to Lack of Bacterial Control upon Salmonella ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: Combined Loss of Caspases-1, -11, -12, and -8 (plus RIPK3) Leads to Lack of Bacterial Control upon Salmonella Infection (A) Bacterial replication over time in WT and Casp1 –/– ;Casp11 –/– mice infected with Salmonella Δ AroA (200 CFU). n = 10−22 mice per group per time point. Mean and SEM are shown. ∗∗ p < 0.005, ∗ p < 0.05, ns p > 0.05 = not significant. (B) Bacterial loads in spleen and liver of mice of the indicated genotypes 3 weeks post-infection with Salmonella Δ AroA (200 CFU). n = 7−48 mice per genotype. Mean and SEM are shown. ∗∗ p < 0.005, ∗ p < 0.05, ns p > 0.05 = not significant. (C) Bacterial loads in spleen and liver from mice of the indicated genotypes 1 to 3 weeks post-infection with Salmonella Δ AroA (200 CFU). n = 3−4 mice per genotype and time point. Mean and SEM are shown. ∗∗ p < 0.005, ∗ p < 0.05, ns p > 0.05 = not significant. (D) Mouse survival curves and corresponding bacterial loads in the spleen and liver at time of sacrifice in WT and Casp1 –/– ;Casp11 –/– ;Casp12 –/– ;Casp8 –/– ;Ripk3 −/− mice infected with Salmonella Δ AroA (200 CFU). n = 7−8 mice per genotype. Mean and SEM are shown. ∗∗ p < 0.005. (E) Bone marrow chimeras of the indicated genotypes were infected with Salmonella Δ AroA (200 CFU) and culled for analysis of bacterial loads in spleen and liver 3 weeks post-infection. n = 10 mice per group. Mean and SEM are shown. ∗∗ p < 0.005. Please also see Figure S1 .

    Techniques Used: Infection

    Caspase-11 Can Compensate for the Loss of Caspases-1 and -8 to Ensure GSDMD-Mediated Killing of Salmonella -Infected Cells (A) LDH release cell death assay of Salmonella SL1344 (MOI = 50) -infected iBMDMs of the indicated genotypes that had been left untreated or treated with VX-765 or Emricasan. Data pooled from two or more experiments. Mean and SEM are shown. ∗∗ p < 0.005, ns p > 0.05 = not significant. (B) LDH release cell death assays of iBMDMs of the indicated genotypes that had been infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ∗∗ p < 0.005, ns p > 0.05 = not significant. (C) LDH release cell death assays of Salmonella -infected iBMDMs of the indicated genotypes or Casp1 –/– ;Casp8 –/– ;Ripk3 –/– iBMDMs that had been left untreated or treated with Emricasan and infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ∗∗ p < 0.005, ns p > 0.05 = not significant. (D) Immunoblot analysis of caspase-11, GSDMD, BID, and PARP in Casp1 –/– ;Casp8 –/– ;Ripk3 –/– iBMDMs that had been left untreated or treated with Emricasan and infected with Salmonella SL1344 (MOI = 50). WT iBMDMs that had been left untreated or treated with LPS for 4 h were used as a control for the induction of caspase-11. Probing for β-actin served as a loading control. Please see also <xref ref-type=Figure S4 . " title="... the indicated genotypes or Casp1 –/– ;Casp8 –/– ;Ripk3 –/– iBMDMs that had been left untreated or ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: Caspase-11 Can Compensate for the Loss of Caspases-1 and -8 to Ensure GSDMD-Mediated Killing of Salmonella -Infected Cells (A) LDH release cell death assay of Salmonella SL1344 (MOI = 50) -infected iBMDMs of the indicated genotypes that had been left untreated or treated with VX-765 or Emricasan. Data pooled from two or more experiments. Mean and SEM are shown. ∗∗ p < 0.005, ns p > 0.05 = not significant. (B) LDH release cell death assays of iBMDMs of the indicated genotypes that had been infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ∗∗ p < 0.005, ns p > 0.05 = not significant. (C) LDH release cell death assays of Salmonella -infected iBMDMs of the indicated genotypes or Casp1 –/– ;Casp8 –/– ;Ripk3 –/– iBMDMs that had been left untreated or treated with Emricasan and infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ∗∗ p < 0.005, ns p > 0.05 = not significant. (D) Immunoblot analysis of caspase-11, GSDMD, BID, and PARP in Casp1 –/– ;Casp8 –/– ;Ripk3 –/– iBMDMs that had been left untreated or treated with Emricasan and infected with Salmonella SL1344 (MOI = 50). WT iBMDMs that had been left untreated or treated with LPS for 4 h were used as a control for the induction of caspase-11. Probing for β-actin served as a loading control. Please see also Figure S4 .

    Techniques Used: Infection, Western Blot

    Caspase-1 Can Activate Caspases-3, -7, and -9 Independently of Caspase-8 and BID (A) LDH release cell death assays of iBMDMs of the indicated genotypes that had been infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ∗∗ p < 0.005, ns p > 0.05 = not significant. (B) iBMDMs of the indicated genotypes were infected with Salmonella SL1344 (MOI = 50) and cleavage associated with activation of the indicated cell death proteins was analyzed by immunoblotting at the indicated time points. Probing for β-actin served as a loading control. (C) LDH release cell death assays of iBMDMs of the indicated genotypes that had been infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ∗∗ p < 0.005, ns p > 0.05 = not significant. (D) GsdmD –/– ;Casp8 –/– ;Ripk3 –/– ;Mlkl –/– ;Casp3 –/– ;Casp7 –/– iBMDMs were infected with Salmonella SL1344 (MOI = 50) and expression and cleavage associated with activation of the indicated cell death proteins was analyzed by immunoblotting at the indicated time points. Probing for β-actin served as a loading control. (E) LDH release cell death assays of iBMDMs of the indicated genotypes that had been infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ns p > 0.05 = not significant. (F) LDH release cell death assays of iBMDMs of the indicated genotypes that had been infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ns p > 0.05 = not significant. Please see also <xref ref-type=Figure S5 . " title="... = not significant. (D) GsdmD –/– ;Casp8 –/– ;Ripk3 –/– ;Mlkl –/– ;Casp3 –/– ;Casp7 –/– iBMDMs ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: Caspase-1 Can Activate Caspases-3, -7, and -9 Independently of Caspase-8 and BID (A) LDH release cell death assays of iBMDMs of the indicated genotypes that had been infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ∗∗ p < 0.005, ns p > 0.05 = not significant. (B) iBMDMs of the indicated genotypes were infected with Salmonella SL1344 (MOI = 50) and cleavage associated with activation of the indicated cell death proteins was analyzed by immunoblotting at the indicated time points. Probing for β-actin served as a loading control. (C) LDH release cell death assays of iBMDMs of the indicated genotypes that had been infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ∗∗ p < 0.005, ns p > 0.05 = not significant. (D) GsdmD –/– ;Casp8 –/– ;Ripk3 –/– ;Mlkl –/– ;Casp3 –/– ;Casp7 –/– iBMDMs were infected with Salmonella SL1344 (MOI = 50) and expression and cleavage associated with activation of the indicated cell death proteins was analyzed by immunoblotting at the indicated time points. Probing for β-actin served as a loading control. (E) LDH release cell death assays of iBMDMs of the indicated genotypes that had been infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ns p > 0.05 = not significant. (F) LDH release cell death assays of iBMDMs of the indicated genotypes that had been infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ns p > 0.05 = not significant. Please see also Figure S5 .

    Techniques Used: Infection, Activation Assay, Western Blot, Expressing

    CRISPR Screen Reveals a Central Role for Caspase-1 in Mediating Salmonella -Infection-Induced Cell Death Independent of All Known Downstream Effectors of Cell Killing (A) LDH release cell death assays of iBMDMs of the indicated genotypes that had been infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ∗ p < 0.05, ns p > 0.05 = not significant. (B) GsdmD –/– ;Bid –/– ;Mlkl –/– ;Casp3 –/– ;Casp7 –/– ;Casp9 –/– iBMDM whole genome CRISPR-Cas9 screen mean-difference (MD) plot showing log-fold change versus average log counts per million (CPM) after three rounds of infection with Salmonella SL1344 (MOI = 50) (please also see A and S5B). (C) GsdmD –/– ;Bid –/– ;Mlkl –/– ;Casp3 –/– ;Casp7 –/– ;Casp9 –/– iBMDMs were infected with Salmonella SL1344 (MOI = 50) and cleavage associated with activation of caspases-1 and -8 was analyzed by immunoblotting at the indicated time points. Probing for β-actin served as a loading control. (D) LDH release cell death assays of WT, Casp1 –/– ;Casp11 –/– ;Casp12 –/– ;Casp8 –/– ;Ripk3 –/– , and two independent clones (#1 and #2) of GsdmD –/– ;Bid –/– ;Mlkl –/– ;Casp3 –/– ;Casp7 –/– ;Casp9 –/– ;Casp1 –/– iBMDMs that had been infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ns p > 0.05 = not significant. Please see also <xref ref-type=Figure S6 . " title="... Casp1 –/– ;Casp11 –/– ;Casp12 –/– ;Casp8 –/– ;Ripk3 –/– , and two independent clones (#1 and ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: CRISPR Screen Reveals a Central Role for Caspase-1 in Mediating Salmonella -Infection-Induced Cell Death Independent of All Known Downstream Effectors of Cell Killing (A) LDH release cell death assays of iBMDMs of the indicated genotypes that had been infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ∗ p < 0.05, ns p > 0.05 = not significant. (B) GsdmD –/– ;Bid –/– ;Mlkl –/– ;Casp3 –/– ;Casp7 –/– ;Casp9 –/– iBMDM whole genome CRISPR-Cas9 screen mean-difference (MD) plot showing log-fold change versus average log counts per million (CPM) after three rounds of infection with Salmonella SL1344 (MOI = 50) (please also see A and S5B). (C) GsdmD –/– ;Bid –/– ;Mlkl –/– ;Casp3 –/– ;Casp7 –/– ;Casp9 –/– iBMDMs were infected with Salmonella SL1344 (MOI = 50) and cleavage associated with activation of caspases-1 and -8 was analyzed by immunoblotting at the indicated time points. Probing for β-actin served as a loading control. (D) LDH release cell death assays of WT, Casp1 –/– ;Casp11 –/– ;Casp12 –/– ;Casp8 –/– ;Ripk3 –/– , and two independent clones (#1 and #2) of GsdmD –/– ;Bid –/– ;Mlkl –/– ;Casp3 –/– ;Casp7 –/– ;Casp9 –/– ;Casp1 –/– iBMDMs that had been infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ns p > 0.05 = not significant. Please see also Figure S6 .

    Techniques Used: CRISPR, Infection, Activation Assay, Western Blot, Clone Assay

    Caspase-1 Can Act Upstream of and Requires Caspase-8 to Induce Cell Death in the Absence of All Known Downstream Effectors of Pyroptosis and Apoptosis (A) iBMDMs of the indicated genotypes were infected with Samonella SL1344 (MOI = 50) and cleavage associated with activation of caspases-1 and -8 was analyzed by immunoblotting at the indicated time points. Probing for HSP70 served as loading control. (B) LDH release death assays of Salmonella SL1344 (MOI = 50) -infected Casp1 –/– ;Casp11 –/– ;Casp12 –/– ;Casp8 –/– ;Ripk3 –/– and GsdmD –/– ;Bid –/– ;Mlkl –/– ;Casp3 –/– ;Casp7 –/– ;Casp9 –/– iBMDMs that had been left untreated or treated with VX-765 or Emricasan. Data pooled from two experiments. Mean and SEM are shown. ∗∗ p < 0.005, ns p > 0.05 = not significant. (C) Immunoblot analysis of caspases-1 and -8 activation at the indicated time points in Salmonella SL1344 (MOI = 50) -infected GsdmD –/– ;Bid –/– ;Mlkl –/– ;Casp3 –/– ;7 –/– ;9 –/– iBMDMs that had been left untreated or treated with VX-765. Probing for HSP70 served as a loading control. Please see also <xref ref-type=Figure S6 . " title="... Casp1 –/– ;Casp11 –/– ;Casp12 –/– ;Casp8 –/– ;Ripk3 –/– and GsdmD –/– ;Bid –/– ;Mlkl –/– ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: Caspase-1 Can Act Upstream of and Requires Caspase-8 to Induce Cell Death in the Absence of All Known Downstream Effectors of Pyroptosis and Apoptosis (A) iBMDMs of the indicated genotypes were infected with Samonella SL1344 (MOI = 50) and cleavage associated with activation of caspases-1 and -8 was analyzed by immunoblotting at the indicated time points. Probing for HSP70 served as loading control. (B) LDH release death assays of Salmonella SL1344 (MOI = 50) -infected Casp1 –/– ;Casp11 –/– ;Casp12 –/– ;Casp8 –/– ;Ripk3 –/– and GsdmD –/– ;Bid –/– ;Mlkl –/– ;Casp3 –/– ;Casp7 –/– ;Casp9 –/– iBMDMs that had been left untreated or treated with VX-765 or Emricasan. Data pooled from two experiments. Mean and SEM are shown. ∗∗ p < 0.005, ns p > 0.05 = not significant. (C) Immunoblot analysis of caspases-1 and -8 activation at the indicated time points in Salmonella SL1344 (MOI = 50) -infected GsdmD –/– ;Bid –/– ;Mlkl –/– ;Casp3 –/– ;7 –/– ;9 –/– iBMDMs that had been left untreated or treated with VX-765. Probing for HSP70 served as a loading control. Please see also Figure S6 .

    Techniques Used: Infection, Activation Assay, Western Blot


    Figure Legend Snippet:

    Techniques Used: Recombinant, Protease Inhibitor, Western Blot, Lactate Dehydrogenase Assay, Plasmid Preparation, Sequencing, Software

    rabbit anti ripk3  (ProSci Incorporated)


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    ProSci Incorporated rabbit anti ripk3
    Combined Loss of Caspases-1, -11, -12, and -8 (plus <t>RIPK3)</t> Leads to Lack of Bacterial Control upon Salmonella Infection (A) Bacterial replication over time in WT and Casp1 –/– ;Casp11 –/– mice infected with Salmonella Δ AroA (200 CFU). n = 10−22 mice per group per time point. Mean and SEM are shown. ∗∗ p < 0.005, ∗ p < 0.05, ns p > 0.05 = not significant. (B) Bacterial loads in spleen and liver of mice of the indicated genotypes 3 weeks post-infection with Salmonella Δ AroA (200 CFU). n = 7−48 mice per genotype. Mean and SEM are shown. ∗∗ p < 0.005, ∗ p < 0.05, ns p > 0.05 = not significant. (C) Bacterial loads in spleen and liver from mice of the indicated genotypes 1 to 3 weeks post-infection with Salmonella Δ AroA (200 CFU). n = 3−4 mice per genotype and time point. Mean and SEM are shown. ∗∗ p < 0.005, ∗ p < 0.05, ns p > 0.05 = not significant. (D) Mouse survival curves and corresponding bacterial loads in the spleen and liver at time of sacrifice in WT and Casp1 –/– ;Casp11 –/– ;Casp12 –/– ;Casp8 –/– ;Ripk3 −/− mice infected with Salmonella Δ AroA (200 CFU). n = 7−8 mice per genotype. Mean and SEM are shown. ∗∗ p < 0.005. (E) Bone marrow chimeras of the indicated genotypes were infected with Salmonella Δ AroA (200 CFU) and culled for analysis of bacterial loads in spleen and liver 3 weeks post-infection. n = 10 mice per group. Mean and SEM are shown. ∗∗ p < 0.005. Please also see <xref ref-type=Figure S1 . " width="250" height="auto" />
    Rabbit Anti Ripk3, supplied by ProSci Incorporated, 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/rabbit anti ripk3/product/ProSci Incorporated
    Average 91 stars, based on 1 article reviews
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    rabbit anti ripk3 - by Bioz Stars, 2023-06
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    1) Product Images from "Flexible Usage and Interconnectivity of Diverse Cell Death Pathways Protect against Intracellular Infection"

    Article Title: Flexible Usage and Interconnectivity of Diverse Cell Death Pathways Protect against Intracellular Infection

    Journal: Immunity

    doi: 10.1016/j.immuni.2020.07.004

    Combined Loss of Caspases-1, -11, -12, and -8 (plus RIPK3) Leads to Lack of Bacterial Control upon Salmonella Infection (A) Bacterial replication over time in WT and Casp1 –/– ;Casp11 –/– mice infected with Salmonella Δ AroA (200 CFU). n = 10−22 mice per group per time point. Mean and SEM are shown. ∗∗ p < 0.005, ∗ p < 0.05, ns p > 0.05 = not significant. (B) Bacterial loads in spleen and liver of mice of the indicated genotypes 3 weeks post-infection with Salmonella Δ AroA (200 CFU). n = 7−48 mice per genotype. Mean and SEM are shown. ∗∗ p < 0.005, ∗ p < 0.05, ns p > 0.05 = not significant. (C) Bacterial loads in spleen and liver from mice of the indicated genotypes 1 to 3 weeks post-infection with Salmonella Δ AroA (200 CFU). n = 3−4 mice per genotype and time point. Mean and SEM are shown. ∗∗ p < 0.005, ∗ p < 0.05, ns p > 0.05 = not significant. (D) Mouse survival curves and corresponding bacterial loads in the spleen and liver at time of sacrifice in WT and Casp1 –/– ;Casp11 –/– ;Casp12 –/– ;Casp8 –/– ;Ripk3 −/− mice infected with Salmonella Δ AroA (200 CFU). n = 7−8 mice per genotype. Mean and SEM are shown. ∗∗ p < 0.005. (E) Bone marrow chimeras of the indicated genotypes were infected with Salmonella Δ AroA (200 CFU) and culled for analysis of bacterial loads in spleen and liver 3 weeks post-infection. n = 10 mice per group. Mean and SEM are shown. ∗∗ p < 0.005. Please also see <xref ref-type=Figure S1 . " title="... Loss of Caspases-1, -11, -12, and -8 (plus RIPK3) Leads to Lack of Bacterial Control upon Salmonella ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: Combined Loss of Caspases-1, -11, -12, and -8 (plus RIPK3) Leads to Lack of Bacterial Control upon Salmonella Infection (A) Bacterial replication over time in WT and Casp1 –/– ;Casp11 –/– mice infected with Salmonella Δ AroA (200 CFU). n = 10−22 mice per group per time point. Mean and SEM are shown. ∗∗ p < 0.005, ∗ p < 0.05, ns p > 0.05 = not significant. (B) Bacterial loads in spleen and liver of mice of the indicated genotypes 3 weeks post-infection with Salmonella Δ AroA (200 CFU). n = 7−48 mice per genotype. Mean and SEM are shown. ∗∗ p < 0.005, ∗ p < 0.05, ns p > 0.05 = not significant. (C) Bacterial loads in spleen and liver from mice of the indicated genotypes 1 to 3 weeks post-infection with Salmonella Δ AroA (200 CFU). n = 3−4 mice per genotype and time point. Mean and SEM are shown. ∗∗ p < 0.005, ∗ p < 0.05, ns p > 0.05 = not significant. (D) Mouse survival curves and corresponding bacterial loads in the spleen and liver at time of sacrifice in WT and Casp1 –/– ;Casp11 –/– ;Casp12 –/– ;Casp8 –/– ;Ripk3 −/− mice infected with Salmonella Δ AroA (200 CFU). n = 7−8 mice per genotype. Mean and SEM are shown. ∗∗ p < 0.005. (E) Bone marrow chimeras of the indicated genotypes were infected with Salmonella Δ AroA (200 CFU) and culled for analysis of bacterial loads in spleen and liver 3 weeks post-infection. n = 10 mice per group. Mean and SEM are shown. ∗∗ p < 0.005. Please also see Figure S1 .

    Techniques Used: Infection

    Caspase-11 Can Compensate for the Loss of Caspases-1 and -8 to Ensure GSDMD-Mediated Killing of Salmonella -Infected Cells (A) LDH release cell death assay of Salmonella SL1344 (MOI = 50) -infected iBMDMs of the indicated genotypes that had been left untreated or treated with VX-765 or Emricasan. Data pooled from two or more experiments. Mean and SEM are shown. ∗∗ p < 0.005, ns p > 0.05 = not significant. (B) LDH release cell death assays of iBMDMs of the indicated genotypes that had been infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ∗∗ p < 0.005, ns p > 0.05 = not significant. (C) LDH release cell death assays of Salmonella -infected iBMDMs of the indicated genotypes or Casp1 –/– ;Casp8 –/– ;Ripk3 –/– iBMDMs that had been left untreated or treated with Emricasan and infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ∗∗ p < 0.005, ns p > 0.05 = not significant. (D) Immunoblot analysis of caspase-11, GSDMD, BID, and PARP in Casp1 –/– ;Casp8 –/– ;Ripk3 –/– iBMDMs that had been left untreated or treated with Emricasan and infected with Salmonella SL1344 (MOI = 50). WT iBMDMs that had been left untreated or treated with LPS for 4 h were used as a control for the induction of caspase-11. Probing for β-actin served as a loading control. Please see also <xref ref-type=Figure S4 . " title="... the indicated genotypes or Casp1 –/– ;Casp8 –/– ;Ripk3 –/– iBMDMs that had been left untreated or ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: Caspase-11 Can Compensate for the Loss of Caspases-1 and -8 to Ensure GSDMD-Mediated Killing of Salmonella -Infected Cells (A) LDH release cell death assay of Salmonella SL1344 (MOI = 50) -infected iBMDMs of the indicated genotypes that had been left untreated or treated with VX-765 or Emricasan. Data pooled from two or more experiments. Mean and SEM are shown. ∗∗ p < 0.005, ns p > 0.05 = not significant. (B) LDH release cell death assays of iBMDMs of the indicated genotypes that had been infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ∗∗ p < 0.005, ns p > 0.05 = not significant. (C) LDH release cell death assays of Salmonella -infected iBMDMs of the indicated genotypes or Casp1 –/– ;Casp8 –/– ;Ripk3 –/– iBMDMs that had been left untreated or treated with Emricasan and infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ∗∗ p < 0.005, ns p > 0.05 = not significant. (D) Immunoblot analysis of caspase-11, GSDMD, BID, and PARP in Casp1 –/– ;Casp8 –/– ;Ripk3 –/– iBMDMs that had been left untreated or treated with Emricasan and infected with Salmonella SL1344 (MOI = 50). WT iBMDMs that had been left untreated or treated with LPS for 4 h were used as a control for the induction of caspase-11. Probing for β-actin served as a loading control. Please see also Figure S4 .

    Techniques Used: Infection, Western Blot

    Caspase-1 Can Activate Caspases-3, -7, and -9 Independently of Caspase-8 and BID (A) LDH release cell death assays of iBMDMs of the indicated genotypes that had been infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ∗∗ p < 0.005, ns p > 0.05 = not significant. (B) iBMDMs of the indicated genotypes were infected with Salmonella SL1344 (MOI = 50) and cleavage associated with activation of the indicated cell death proteins was analyzed by immunoblotting at the indicated time points. Probing for β-actin served as a loading control. (C) LDH release cell death assays of iBMDMs of the indicated genotypes that had been infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ∗∗ p < 0.005, ns p > 0.05 = not significant. (D) GsdmD –/– ;Casp8 –/– ;Ripk3 –/– ;Mlkl –/– ;Casp3 –/– ;Casp7 –/– iBMDMs were infected with Salmonella SL1344 (MOI = 50) and expression and cleavage associated with activation of the indicated cell death proteins was analyzed by immunoblotting at the indicated time points. Probing for β-actin served as a loading control. (E) LDH release cell death assays of iBMDMs of the indicated genotypes that had been infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ns p > 0.05 = not significant. (F) LDH release cell death assays of iBMDMs of the indicated genotypes that had been infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ns p > 0.05 = not significant. Please see also <xref ref-type=Figure S5 . " title="... = not significant. (D) GsdmD –/– ;Casp8 –/– ;Ripk3 –/– ;Mlkl –/– ;Casp3 –/– ;Casp7 –/– iBMDMs ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: Caspase-1 Can Activate Caspases-3, -7, and -9 Independently of Caspase-8 and BID (A) LDH release cell death assays of iBMDMs of the indicated genotypes that had been infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ∗∗ p < 0.005, ns p > 0.05 = not significant. (B) iBMDMs of the indicated genotypes were infected with Salmonella SL1344 (MOI = 50) and cleavage associated with activation of the indicated cell death proteins was analyzed by immunoblotting at the indicated time points. Probing for β-actin served as a loading control. (C) LDH release cell death assays of iBMDMs of the indicated genotypes that had been infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ∗∗ p < 0.005, ns p > 0.05 = not significant. (D) GsdmD –/– ;Casp8 –/– ;Ripk3 –/– ;Mlkl –/– ;Casp3 –/– ;Casp7 –/– iBMDMs were infected with Salmonella SL1344 (MOI = 50) and expression and cleavage associated with activation of the indicated cell death proteins was analyzed by immunoblotting at the indicated time points. Probing for β-actin served as a loading control. (E) LDH release cell death assays of iBMDMs of the indicated genotypes that had been infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ns p > 0.05 = not significant. (F) LDH release cell death assays of iBMDMs of the indicated genotypes that had been infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ns p > 0.05 = not significant. Please see also Figure S5 .

    Techniques Used: Infection, Activation Assay, Western Blot, Expressing

    CRISPR Screen Reveals a Central Role for Caspase-1 in Mediating Salmonella -Infection-Induced Cell Death Independent of All Known Downstream Effectors of Cell Killing (A) LDH release cell death assays of iBMDMs of the indicated genotypes that had been infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ∗ p < 0.05, ns p > 0.05 = not significant. (B) GsdmD –/– ;Bid –/– ;Mlkl –/– ;Casp3 –/– ;Casp7 –/– ;Casp9 –/– iBMDM whole genome CRISPR-Cas9 screen mean-difference (MD) plot showing log-fold change versus average log counts per million (CPM) after three rounds of infection with Salmonella SL1344 (MOI = 50) (please also see A and S5B). (C) GsdmD –/– ;Bid –/– ;Mlkl –/– ;Casp3 –/– ;Casp7 –/– ;Casp9 –/– iBMDMs were infected with Salmonella SL1344 (MOI = 50) and cleavage associated with activation of caspases-1 and -8 was analyzed by immunoblotting at the indicated time points. Probing for β-actin served as a loading control. (D) LDH release cell death assays of WT, Casp1 –/– ;Casp11 –/– ;Casp12 –/– ;Casp8 –/– ;Ripk3 –/– , and two independent clones (#1 and #2) of GsdmD –/– ;Bid –/– ;Mlkl –/– ;Casp3 –/– ;Casp7 –/– ;Casp9 –/– ;Casp1 –/– iBMDMs that had been infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ns p > 0.05 = not significant. Please see also <xref ref-type=Figure S6 . " title="... Casp1 –/– ;Casp11 –/– ;Casp12 –/– ;Casp8 –/– ;Ripk3 –/– , and two independent clones (#1 and ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: CRISPR Screen Reveals a Central Role for Caspase-1 in Mediating Salmonella -Infection-Induced Cell Death Independent of All Known Downstream Effectors of Cell Killing (A) LDH release cell death assays of iBMDMs of the indicated genotypes that had been infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ∗ p < 0.05, ns p > 0.05 = not significant. (B) GsdmD –/– ;Bid –/– ;Mlkl –/– ;Casp3 –/– ;Casp7 –/– ;Casp9 –/– iBMDM whole genome CRISPR-Cas9 screen mean-difference (MD) plot showing log-fold change versus average log counts per million (CPM) after three rounds of infection with Salmonella SL1344 (MOI = 50) (please also see A and S5B). (C) GsdmD –/– ;Bid –/– ;Mlkl –/– ;Casp3 –/– ;Casp7 –/– ;Casp9 –/– iBMDMs were infected with Salmonella SL1344 (MOI = 50) and cleavage associated with activation of caspases-1 and -8 was analyzed by immunoblotting at the indicated time points. Probing for β-actin served as a loading control. (D) LDH release cell death assays of WT, Casp1 –/– ;Casp11 –/– ;Casp12 –/– ;Casp8 –/– ;Ripk3 –/– , and two independent clones (#1 and #2) of GsdmD –/– ;Bid –/– ;Mlkl –/– ;Casp3 –/– ;Casp7 –/– ;Casp9 –/– ;Casp1 –/– iBMDMs that had been infected with Salmonella SL1344 (MOI = 50). Data pooled from two or more experiments. Mean and SEM are shown. ns p > 0.05 = not significant. Please see also Figure S6 .

    Techniques Used: CRISPR, Infection, Activation Assay, Western Blot, Clone Assay

    Caspase-1 Can Act Upstream of and Requires Caspase-8 to Induce Cell Death in the Absence of All Known Downstream Effectors of Pyroptosis and Apoptosis (A) iBMDMs of the indicated genotypes were infected with Samonella SL1344 (MOI = 50) and cleavage associated with activation of caspases-1 and -8 was analyzed by immunoblotting at the indicated time points. Probing for HSP70 served as loading control. (B) LDH release death assays of Salmonella SL1344 (MOI = 50) -infected Casp1 –/– ;Casp11 –/– ;Casp12 –/– ;Casp8 –/– ;Ripk3 –/– and GsdmD –/– ;Bid –/– ;Mlkl –/– ;Casp3 –/– ;Casp7 –/– ;Casp9 –/– iBMDMs that had been left untreated or treated with VX-765 or Emricasan. Data pooled from two experiments. Mean and SEM are shown. ∗∗ p < 0.005, ns p > 0.05 = not significant. (C) Immunoblot analysis of caspases-1 and -8 activation at the indicated time points in Salmonella SL1344 (MOI = 50) -infected GsdmD –/– ;Bid –/– ;Mlkl –/– ;Casp3 –/– ;7 –/– ;9 –/– iBMDMs that had been left untreated or treated with VX-765. Probing for HSP70 served as a loading control. Please see also <xref ref-type=Figure S6 . " title="... Casp1 –/– ;Casp11 –/– ;Casp12 –/– ;Casp8 –/– ;Ripk3 –/– and GsdmD –/– ;Bid –/– ;Mlkl –/– ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: Caspase-1 Can Act Upstream of and Requires Caspase-8 to Induce Cell Death in the Absence of All Known Downstream Effectors of Pyroptosis and Apoptosis (A) iBMDMs of the indicated genotypes were infected with Samonella SL1344 (MOI = 50) and cleavage associated with activation of caspases-1 and -8 was analyzed by immunoblotting at the indicated time points. Probing for HSP70 served as loading control. (B) LDH release death assays of Salmonella SL1344 (MOI = 50) -infected Casp1 –/– ;Casp11 –/– ;Casp12 –/– ;Casp8 –/– ;Ripk3 –/– and GsdmD –/– ;Bid –/– ;Mlkl –/– ;Casp3 –/– ;Casp7 –/– ;Casp9 –/– iBMDMs that had been left untreated or treated with VX-765 or Emricasan. Data pooled from two experiments. Mean and SEM are shown. ∗∗ p < 0.005, ns p > 0.05 = not significant. (C) Immunoblot analysis of caspases-1 and -8 activation at the indicated time points in Salmonella SL1344 (MOI = 50) -infected GsdmD –/– ;Bid –/– ;Mlkl –/– ;Casp3 –/– ;7 –/– ;9 –/– iBMDMs that had been left untreated or treated with VX-765. Probing for HSP70 served as a loading control. Please see also Figure S6 .

    Techniques Used: Infection, Activation Assay, Western Blot


    Figure Legend Snippet:

    Techniques Used: Recombinant, Protease Inhibitor, Western Blot, Lactate Dehydrogenase Assay, Plasmid Preparation, Sequencing, Software

    rabbit anti ripk3  (ProSci Incorporated)


    Bioz Verified Symbol ProSci Incorporated is a verified supplier
    Bioz Manufacturer Symbol ProSci Incorporated manufactures this product  
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    • 92
      Buy from Supplier

    Structured Review

    ProSci Incorporated rabbit anti ripk3
    Rabbit Anti Ripk3, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rabbit anti ripk3  (ProSci Incorporated)


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    ProSci Incorporated rabbit anti ripk3
    a – c Kinetics of cell death of WT MEFs ( a ), <t>Ripk3</t> −/− MEFs ( b ), and Mlkl − /− MEFs ( c ) infected with HSV1 (F or KOS) or HSV1 mutant virus, respectively, at a multiplicity of infection, measured in real time by Sytox Green incorporation. d Micrograph images of WT MEFs and Zbp1 −/− MEFs infected with HSV1 (F) or HSV1 (F mut RHIM) for 24 h in the presence of Sytox Green. Scale bar = 200 μm. e Kinetics of cell death of Zbp1 −/− MEFs infected with HSV1 (F or KOS) or HSV1 ICP6 mutant viruses, respectively, measured in real time by Sytox Green incorporation. f Cell viability of WT, Zbp1 − /− , and RIPK3 kinase inactive (RIPK3K51A) MEFs infected with HSV1 (F) or HSV1 (F mut RHIM). g Kinetics of cell death of SVEC4-10 cells infected with HSV1 (F) or HSV1 (F mut RHIM), measured in real time by Sytox Green incorporation. h Kinetics of cell death of ZBP1-deficient SVEC4-10 cells following infection with HSV1 (F) or HSV1 (F mut RHIM). ZBP1 expression was confirmed by IB (right). Black solid triangle represents F, black solid square represents KOS, hollow triangle represents F mut RHIM, hollow square represents ∆ICP6, and solid circle represents Mock. hpi hours post infection. See also Supplemental Fig.
    Rabbit Anti Ripk3, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Species-independent contribution of ZBP1/DAI/DLM-1-triggered necroptosis in host defense against HSV1"

    Article Title: Species-independent contribution of ZBP1/DAI/DLM-1-triggered necroptosis in host defense against HSV1

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-0868-3

    a – c Kinetics of cell death of WT MEFs ( a ), Ripk3 −/− MEFs ( b ), and Mlkl − /− MEFs ( c ) infected with HSV1 (F or KOS) or HSV1 mutant virus, respectively, at a multiplicity of infection, measured in real time by Sytox Green incorporation. d Micrograph images of WT MEFs and Zbp1 −/− MEFs infected with HSV1 (F) or HSV1 (F mut RHIM) for 24 h in the presence of Sytox Green. Scale bar = 200 μm. e Kinetics of cell death of Zbp1 −/− MEFs infected with HSV1 (F or KOS) or HSV1 ICP6 mutant viruses, respectively, measured in real time by Sytox Green incorporation. f Cell viability of WT, Zbp1 − /− , and RIPK3 kinase inactive (RIPK3K51A) MEFs infected with HSV1 (F) or HSV1 (F mut RHIM). g Kinetics of cell death of SVEC4-10 cells infected with HSV1 (F) or HSV1 (F mut RHIM), measured in real time by Sytox Green incorporation. h Kinetics of cell death of ZBP1-deficient SVEC4-10 cells following infection with HSV1 (F) or HSV1 (F mut RHIM). ZBP1 expression was confirmed by IB (right). Black solid triangle represents F, black solid square represents KOS, hollow triangle represents F mut RHIM, hollow square represents ∆ICP6, and solid circle represents Mock. hpi hours post infection. See also Supplemental Fig.
    Figure Legend Snippet: a – c Kinetics of cell death of WT MEFs ( a ), Ripk3 −/− MEFs ( b ), and Mlkl − /− MEFs ( c ) infected with HSV1 (F or KOS) or HSV1 mutant virus, respectively, at a multiplicity of infection, measured in real time by Sytox Green incorporation. d Micrograph images of WT MEFs and Zbp1 −/− MEFs infected with HSV1 (F) or HSV1 (F mut RHIM) for 24 h in the presence of Sytox Green. Scale bar = 200 μm. e Kinetics of cell death of Zbp1 −/− MEFs infected with HSV1 (F or KOS) or HSV1 ICP6 mutant viruses, respectively, measured in real time by Sytox Green incorporation. f Cell viability of WT, Zbp1 − /− , and RIPK3 kinase inactive (RIPK3K51A) MEFs infected with HSV1 (F) or HSV1 (F mut RHIM). g Kinetics of cell death of SVEC4-10 cells infected with HSV1 (F) or HSV1 (F mut RHIM), measured in real time by Sytox Green incorporation. h Kinetics of cell death of ZBP1-deficient SVEC4-10 cells following infection with HSV1 (F) or HSV1 (F mut RHIM). ZBP1 expression was confirmed by IB (right). Black solid triangle represents F, black solid square represents KOS, hollow triangle represents F mut RHIM, hollow square represents ∆ICP6, and solid circle represents Mock. hpi hours post infection. See also Supplemental Fig.

    Techniques Used: Infection, Mutagenesis, Expressing

    a , b Viral titers of WT MEF and Zbp1 −/− MEF cells infected with HSV1(F mut RHIM) viruses at ( a ) MOI = 5 or ( b ) MOI = 0.1 for indicated times. Cells together with the supernatants were harvested and titered by a standard viral plaque assay. pfu plaque-forming units. c Viral titers of spleen of WT, Zbp1 −/ − , Ripk3 −/− , and Mlkl −/− mice infected with 1 × 10 7 pfu HSV1 (FmutRHIM) per mouse via intraperitoneal inoculation (i.p.). One-way ANOVA multiple comparison post-test analyses were conducted using GraphPad Prism 5. p < 0.05 was considered significant
    Figure Legend Snippet: a , b Viral titers of WT MEF and Zbp1 −/− MEF cells infected with HSV1(F mut RHIM) viruses at ( a ) MOI = 5 or ( b ) MOI = 0.1 for indicated times. Cells together with the supernatants were harvested and titered by a standard viral plaque assay. pfu plaque-forming units. c Viral titers of spleen of WT, Zbp1 −/ − , Ripk3 −/− , and Mlkl −/− mice infected with 1 × 10 7 pfu HSV1 (FmutRHIM) per mouse via intraperitoneal inoculation (i.p.). One-way ANOVA multiple comparison post-test analyses were conducted using GraphPad Prism 5. p < 0.05 was considered significant

    Techniques Used: Infection, Viral Plaque Assay

    a , b IB analysis to detect p-MLKL, total MLKL, ZBP1, ICP0, and β-actin from WT MEFs ( a ) or SVEC4-10 ( b ) infected with HSV1 (F) or HSV1 (F mut RHIM) for the indicated times. c Immunoprecipitation (IP) analysis to detect the interaction of ZBP1 and RIPK3 in SVEC4-10 cells. Wild-type SVEC4-10 cells were infected with HSV1 (F mut RHIM), mock infected cells as the control. ZBP1-deficient SVEC4-10 cells infected with the same amount HSV1 (F mut RHIM) were also employed as a control. RIPK3 were immunoprecipitated, and ZBP1, RIPK3, and MLKL were analyzed by IB. Whole-cell extract (5% input) was examined in parallel for RIPK3, ZBP1, MLKL, and HSV1 proteins-ICP0. Black solid triangle represents F, hollow triangle represents F mut RHIM, and solid circular represents mock. d Kinetics of cell death of type I interferon receptor (IFNAR)-deficient MEFs infected with HSV1 (F) or HSV1 (F mut RHIM). See also Supplementary Figure
    Figure Legend Snippet: a , b IB analysis to detect p-MLKL, total MLKL, ZBP1, ICP0, and β-actin from WT MEFs ( a ) or SVEC4-10 ( b ) infected with HSV1 (F) or HSV1 (F mut RHIM) for the indicated times. c Immunoprecipitation (IP) analysis to detect the interaction of ZBP1 and RIPK3 in SVEC4-10 cells. Wild-type SVEC4-10 cells were infected with HSV1 (F mut RHIM), mock infected cells as the control. ZBP1-deficient SVEC4-10 cells infected with the same amount HSV1 (F mut RHIM) were also employed as a control. RIPK3 were immunoprecipitated, and ZBP1, RIPK3, and MLKL were analyzed by IB. Whole-cell extract (5% input) was examined in parallel for RIPK3, ZBP1, MLKL, and HSV1 proteins-ICP0. Black solid triangle represents F, hollow triangle represents F mut RHIM, and solid circular represents mock. d Kinetics of cell death of type I interferon receptor (IFNAR)-deficient MEFs infected with HSV1 (F) or HSV1 (F mut RHIM). See also Supplementary Figure

    Techniques Used: Infection, Immunoprecipitation

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    • rabbit anti ripk3  (ProSci Incorporated)
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    ProSci Incorporated rabbit anti ripk3
    A , B U937 cell lines of various genotypes expressing an inducible MLKL(1-180)-gyrase fusion were treated with doxycycline (to induce expression) and/or coumermycin (to induce dimerisation), or left untreated. After 24 h ( A ) or 48 h ( B ), cells were harvested and assessed for cell death (PI uptake measured using flow cytometry), counting a minimum of 5000 cells. Data represent three independent experiments, with the exception of the BAX −/− BAK −/− cell lines, where n = 2. Statistics were calculated in GraphPad Prism 8, and p values are shown where <0.05. C The same U937 cells used in panel A and B were treated with doxycycline alone and left overnight. After approximately 16 h, cells were harvested and lysed for analysis of fusion protein expression via western blot. The upper MLKL band (*) represents endogenous MLKL, and the lower band (arrow) represents the MLKL (1-180) -gyrase fusion protein. D Wild-type U937 were treated with TNF (100 ng/ml), Smac-mimetic (0.5 µM), and caspase inhibitor IDN-6556 (emricasan; 5 µM) (TSI) to induce necroptosis or TNF and Smac-mimetic (TS) alone to induce apoptosis. In parallel, MLKL −/− U937 expressing an inducible MLKL (1-180) -gyrase fusion were treated with doxycycline and coumermycin in the presence or absence of necrosulfonamide (NSA). Images were taken at the specified timepoints using an IncuCyte S3 System to track morphological changes and PI uptake (shown by red). Cells with a necroptotic phenotype (cellular swelling) are indicate with yellow arrows. Three fields were examined per well with representative data shown. E Wild-type U937 cells were treated with doxycycline (20 nM) to induce expression of human wild-type <t>RIPK3</t> in the presence of various cell death inhibitors, as indicated, for 24 h. At the conclusion of the experiment, cells were analysed for PI uptake using flow cytometry, with a minimum of 5000 cells counted. Data represent four or more independent experiments. F Wild-type, MLKL −/− and RIPK1 −/− U937 expressing inducible human wild-type RIPK3 were treated with doxycycline (20 nM) and IDN-6556 (5 µM), doxycycline alone, or left untreated. After 24 h, cell death was measured as PI uptake using flow cytometry. Data are a summary of six independent experiments.
    Rabbit Anti Ripk3, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rabbit polyclonal anti ripk3  (ProSci Incorporated)
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    ProSci Incorporated rabbit polyclonal anti ripk3
    (A) IAV-induced cell death kinetics in primary wild-type (WT), Zbp1−/−, and <t>Ripk3−/−</t> MEFs.
    Rabbit Polyclonal Anti Ripk3, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    A , B U937 cell lines of various genotypes expressing an inducible MLKL(1-180)-gyrase fusion were treated with doxycycline (to induce expression) and/or coumermycin (to induce dimerisation), or left untreated. After 24 h ( A ) or 48 h ( B ), cells were harvested and assessed for cell death (PI uptake measured using flow cytometry), counting a minimum of 5000 cells. Data represent three independent experiments, with the exception of the BAX −/− BAK −/− cell lines, where n = 2. Statistics were calculated in GraphPad Prism 8, and p values are shown where <0.05. C The same U937 cells used in panel A and B were treated with doxycycline alone and left overnight. After approximately 16 h, cells were harvested and lysed for analysis of fusion protein expression via western blot. The upper MLKL band (*) represents endogenous MLKL, and the lower band (arrow) represents the MLKL (1-180) -gyrase fusion protein. D Wild-type U937 were treated with TNF (100 ng/ml), Smac-mimetic (0.5 µM), and caspase inhibitor IDN-6556 (emricasan; 5 µM) (TSI) to induce necroptosis or TNF and Smac-mimetic (TS) alone to induce apoptosis. In parallel, MLKL −/− U937 expressing an inducible MLKL (1-180) -gyrase fusion were treated with doxycycline and coumermycin in the presence or absence of necrosulfonamide (NSA). Images were taken at the specified timepoints using an IncuCyte S3 System to track morphological changes and PI uptake (shown by red). Cells with a necroptotic phenotype (cellular swelling) are indicate with yellow arrows. Three fields were examined per well with representative data shown. E Wild-type U937 cells were treated with doxycycline (20 nM) to induce expression of human wild-type RIPK3 in the presence of various cell death inhibitors, as indicated, for 24 h. At the conclusion of the experiment, cells were analysed for PI uptake using flow cytometry, with a minimum of 5000 cells counted. Data represent four or more independent experiments. F Wild-type, MLKL −/− and RIPK1 −/− U937 expressing inducible human wild-type RIPK3 were treated with doxycycline (20 nM) and IDN-6556 (5 µM), doxycycline alone, or left untreated. After 24 h, cell death was measured as PI uptake using flow cytometry. Data are a summary of six independent experiments.

    Journal: Cell Death & Disease

    Article Title: The Lck inhibitor, AMG-47a, blocks necroptosis and implicates RIPK1 in signalling downstream of MLKL

    doi: 10.1038/s41419-022-04740-w

    Figure Lengend Snippet: A , B U937 cell lines of various genotypes expressing an inducible MLKL(1-180)-gyrase fusion were treated with doxycycline (to induce expression) and/or coumermycin (to induce dimerisation), or left untreated. After 24 h ( A ) or 48 h ( B ), cells were harvested and assessed for cell death (PI uptake measured using flow cytometry), counting a minimum of 5000 cells. Data represent three independent experiments, with the exception of the BAX −/− BAK −/− cell lines, where n = 2. Statistics were calculated in GraphPad Prism 8, and p values are shown where <0.05. C The same U937 cells used in panel A and B were treated with doxycycline alone and left overnight. After approximately 16 h, cells were harvested and lysed for analysis of fusion protein expression via western blot. The upper MLKL band (*) represents endogenous MLKL, and the lower band (arrow) represents the MLKL (1-180) -gyrase fusion protein. D Wild-type U937 were treated with TNF (100 ng/ml), Smac-mimetic (0.5 µM), and caspase inhibitor IDN-6556 (emricasan; 5 µM) (TSI) to induce necroptosis or TNF and Smac-mimetic (TS) alone to induce apoptosis. In parallel, MLKL −/− U937 expressing an inducible MLKL (1-180) -gyrase fusion were treated with doxycycline and coumermycin in the presence or absence of necrosulfonamide (NSA). Images were taken at the specified timepoints using an IncuCyte S3 System to track morphological changes and PI uptake (shown by red). Cells with a necroptotic phenotype (cellular swelling) are indicate with yellow arrows. Three fields were examined per well with representative data shown. E Wild-type U937 cells were treated with doxycycline (20 nM) to induce expression of human wild-type RIPK3 in the presence of various cell death inhibitors, as indicated, for 24 h. At the conclusion of the experiment, cells were analysed for PI uptake using flow cytometry, with a minimum of 5000 cells counted. Data represent four or more independent experiments. F Wild-type, MLKL −/− and RIPK1 −/− U937 expressing inducible human wild-type RIPK3 were treated with doxycycline (20 nM) and IDN-6556 (5 µM), doxycycline alone, or left untreated. After 24 h, cell death was measured as PI uptake using flow cytometry. Data are a summary of six independent experiments.

    Article Snippet: The following primary antibodies were used in this study: rat anti-MLKL clone 3H1 (available as MABC604 from Merck Millipore; 1:1000) was produced in-house as previously described [ ]; rabbit anti-human MLKL pS358 (ab187091, Abcam, Cambridge, United Kingdom; 1:4000); rabbit anti-mouse MLKL pS345 (ab196436, Abcam; 1:1000); rat anti-human RIPK3 (clone 1H2; available as MABC1640 from Merck Millipore; 1:1000) and rat anti-mouse RIPK3 (clone 8G7; available as MABC1595 from Merck Millipore; 1:1000) produced in-house as previously described [ , ]; rabbit anti-RIPK3 (2283, ProSci; 1:1000); rabbit anti-human RIPK3 pS227 (ab209384, Abcam; 1:2000); rabbit anti-mouse RIPK3 pT231/pS232 (GEN135-35-9, kindly supplied by Genentech, South San Francisco, CA, USA [ ]; 1:2000); mouse anti-RIPK1 (610458, BD Transduction Laboratories, San Jose, CA, USA; 1:1000); rabbit anti-RIPK1 (3493, Cell Signalling Technology, Danvers, MA, USA; 1:1000); rabbit anti-human RIPK1 pS166 (65746, Cell Signalling Technology; 1:1000); rabbit anti-mouse RIPK1 pS166 (31122, Cell Signalling Technology; 1:1000); rabbit anti-mouse CASP8 (4927, Cell Signalling Technology; 1:1000); rabbit anti-BAX (2772, Cell Signalling Technology; 1:1000); rabbit anti-BAX (06-536, Upstate Biotechnology, Lake Placid, NY, USA; 1:1000); rabbit anti-GAPDH (2118, Cell Signalling Technology; 1:1000); and mouse anti-β-Actin (A1978, Sigma-Aldrich; 1:3000).

    Techniques: Expressing, Flow Cytometry, Western Blot

    A Three different experimental protocols were used to assess the ability of AMG-47a to inhibit cell death caused by the expression and dimerisation of the MLKL (1-180) -gyrase fusion protein. In protocol 1, inhibitors (or DMSO) were added first, followed by doxycycline and coumermycin together, so that the fusion protein could dimerise on expression. In protocol 2, addition of coumermycin was delayed to allow levels of the fusion protein to accumulate before dimerisation was induced. In protocol 3, the addition of inhibitors (or DMSO) was also delayed, so that it was added after the fusion protein had been expressed for 16 h, but before the addition of coumermycin. In all experimental methods, cells were harvested 24 h after the experiment was initiated. B Wild-type U937 cells expressing the MLKL (1-180) -gyrase fusion protein were treated using either protocol 1, 2, or 3, as described above ( A ). At the conclusion of the experiment, cells were analysed for PI uptake using flow cytometry, with a minimum of 5000 cells counted. Data represent three independent experiments; bars indicate the mean and error bars indicate standard error of the mean. Statistics were calculated in GraphPad Prism8, and p values are shown where <0.05. C Wild-type U937 cells with inducible human RIPK3 were treated with increasing doses of AMG-47a in the presence of 40 nM doxycycline, to induce expression, and 5 µM IDN-6556, to block caspase-dependent cell death. After 48 h, cells were harvested and viability assessed using the Cell-Titer Glo 2 (Promega) system. Data represent two independent experiments (four replicates per experiment) and have been normalised against doxycycline plus IDN-6556 (0% viability) and doxycycline plus IDN-6556 plus NSA (100% viability).

    Journal: Cell Death & Disease

    Article Title: The Lck inhibitor, AMG-47a, blocks necroptosis and implicates RIPK1 in signalling downstream of MLKL

    doi: 10.1038/s41419-022-04740-w

    Figure Lengend Snippet: A Three different experimental protocols were used to assess the ability of AMG-47a to inhibit cell death caused by the expression and dimerisation of the MLKL (1-180) -gyrase fusion protein. In protocol 1, inhibitors (or DMSO) were added first, followed by doxycycline and coumermycin together, so that the fusion protein could dimerise on expression. In protocol 2, addition of coumermycin was delayed to allow levels of the fusion protein to accumulate before dimerisation was induced. In protocol 3, the addition of inhibitors (or DMSO) was also delayed, so that it was added after the fusion protein had been expressed for 16 h, but before the addition of coumermycin. In all experimental methods, cells were harvested 24 h after the experiment was initiated. B Wild-type U937 cells expressing the MLKL (1-180) -gyrase fusion protein were treated using either protocol 1, 2, or 3, as described above ( A ). At the conclusion of the experiment, cells were analysed for PI uptake using flow cytometry, with a minimum of 5000 cells counted. Data represent three independent experiments; bars indicate the mean and error bars indicate standard error of the mean. Statistics were calculated in GraphPad Prism8, and p values are shown where <0.05. C Wild-type U937 cells with inducible human RIPK3 were treated with increasing doses of AMG-47a in the presence of 40 nM doxycycline, to induce expression, and 5 µM IDN-6556, to block caspase-dependent cell death. After 48 h, cells were harvested and viability assessed using the Cell-Titer Glo 2 (Promega) system. Data represent two independent experiments (four replicates per experiment) and have been normalised against doxycycline plus IDN-6556 (0% viability) and doxycycline plus IDN-6556 plus NSA (100% viability).

    Article Snippet: The following primary antibodies were used in this study: rat anti-MLKL clone 3H1 (available as MABC604 from Merck Millipore; 1:1000) was produced in-house as previously described [ ]; rabbit anti-human MLKL pS358 (ab187091, Abcam, Cambridge, United Kingdom; 1:4000); rabbit anti-mouse MLKL pS345 (ab196436, Abcam; 1:1000); rat anti-human RIPK3 (clone 1H2; available as MABC1640 from Merck Millipore; 1:1000) and rat anti-mouse RIPK3 (clone 8G7; available as MABC1595 from Merck Millipore; 1:1000) produced in-house as previously described [ , ]; rabbit anti-RIPK3 (2283, ProSci; 1:1000); rabbit anti-human RIPK3 pS227 (ab209384, Abcam; 1:2000); rabbit anti-mouse RIPK3 pT231/pS232 (GEN135-35-9, kindly supplied by Genentech, South San Francisco, CA, USA [ ]; 1:2000); mouse anti-RIPK1 (610458, BD Transduction Laboratories, San Jose, CA, USA; 1:1000); rabbit anti-RIPK1 (3493, Cell Signalling Technology, Danvers, MA, USA; 1:1000); rabbit anti-human RIPK1 pS166 (65746, Cell Signalling Technology; 1:1000); rabbit anti-mouse RIPK1 pS166 (31122, Cell Signalling Technology; 1:1000); rabbit anti-mouse CASP8 (4927, Cell Signalling Technology; 1:1000); rabbit anti-BAX (2772, Cell Signalling Technology; 1:1000); rabbit anti-BAX (06-536, Upstate Biotechnology, Lake Placid, NY, USA; 1:1000); rabbit anti-GAPDH (2118, Cell Signalling Technology; 1:1000); and mouse anti-β-Actin (A1978, Sigma-Aldrich; 1:3000).

    Techniques: Expressing, Flow Cytometry, Blocking Assay

    A, B, C U937, HT29 and MDF cell lines were treated with DMSO, AMG-47a (1 µM for U937 and MDF; 2 µM for HT29), TSI, or a combination of AMG-47a and TSI over a time course as indicated in the individual figure panels. At the conclusion of the experiment, cells were harvested and lysed for western blot analysis. All cell lines were assessed for phosphorylation and total protein of the three key necroptosis effector proteins, RIPK1, RIPK3, and MLKL, and total β-actin was used as a loading control. Where multiple bands are present, the specific band of interest is indicated with a red arrow. Data are representative of at least three independent experiments. U937 ( D ) or MDF ( E ) cell lines were treated with AMG-47a at 10 µM, the RIPK1 inhibitors (GSK-481, Nec-1s) or a RIPK3 inhibitor (GSK-872) at 20 µM, or an equivalent amount of DMSO, for 1 h at 37 °C. Cell suspensions were then heated over an increasing temperature gradient (as indicated in the methods) for 3 min. Cells were then lysed, and the amount of remaining soluble protein at each temperature point was analysed using Western blot. Results are representative of two (RIPK1 and RIPK3 in U937) or three (all other analyses) independent experiments. AMG-47a binding to RIPK1 ( F ) and RIPK3 ( G ) was measured in competitive binding assays using the KINOMEscan ® Assay Platform (DiscoverX). Graphs were plotted using the raw data supplied from DiscoverX, and a curve was fitted using non-linear regression (GraphPad Prism) to determine the K d and associated error (95% confidence interval). Data represent one (RIPK3) or two (RIPK1) independent experiments, with two runs performed in all experiments and each colour on the graph representing a separate run. All data points were used for the non-linear regression. K d determinations for individual replicates can be found in supplementary data (Supplementary Fig. ). H , I AMG-47a was serially diluted from a maximum concentration of 50 µM (RIPK1) or 100 µM (RIPK3), and its ability to inhibit human RIPK1 or RIPK3 kinase activity was assessed using the ADP-Glo Kinase Assay (Promega). The reaction was allowed to proceed for 4 h before readouts were performed, and data were normalised to DMSO (0% inhibition) and either 1 µM GSK-481 (100% inhibition of RIPK1) or 1 µM GSK-872 (100% inhibition of RIPK3). Data are a summary of two independent experiments performed in duplicate, and the IC 50 and associated error (95% confidence interval) were determined using non-linear regression, as above. Each colour on the graph represents one independent experiment.

    Journal: Cell Death & Disease

    Article Title: The Lck inhibitor, AMG-47a, blocks necroptosis and implicates RIPK1 in signalling downstream of MLKL

    doi: 10.1038/s41419-022-04740-w

    Figure Lengend Snippet: A, B, C U937, HT29 and MDF cell lines were treated with DMSO, AMG-47a (1 µM for U937 and MDF; 2 µM for HT29), TSI, or a combination of AMG-47a and TSI over a time course as indicated in the individual figure panels. At the conclusion of the experiment, cells were harvested and lysed for western blot analysis. All cell lines were assessed for phosphorylation and total protein of the three key necroptosis effector proteins, RIPK1, RIPK3, and MLKL, and total β-actin was used as a loading control. Where multiple bands are present, the specific band of interest is indicated with a red arrow. Data are representative of at least three independent experiments. U937 ( D ) or MDF ( E ) cell lines were treated with AMG-47a at 10 µM, the RIPK1 inhibitors (GSK-481, Nec-1s) or a RIPK3 inhibitor (GSK-872) at 20 µM, or an equivalent amount of DMSO, for 1 h at 37 °C. Cell suspensions were then heated over an increasing temperature gradient (as indicated in the methods) for 3 min. Cells were then lysed, and the amount of remaining soluble protein at each temperature point was analysed using Western blot. Results are representative of two (RIPK1 and RIPK3 in U937) or three (all other analyses) independent experiments. AMG-47a binding to RIPK1 ( F ) and RIPK3 ( G ) was measured in competitive binding assays using the KINOMEscan ® Assay Platform (DiscoverX). Graphs were plotted using the raw data supplied from DiscoverX, and a curve was fitted using non-linear regression (GraphPad Prism) to determine the K d and associated error (95% confidence interval). Data represent one (RIPK3) or two (RIPK1) independent experiments, with two runs performed in all experiments and each colour on the graph representing a separate run. All data points were used for the non-linear regression. K d determinations for individual replicates can be found in supplementary data (Supplementary Fig. ). H , I AMG-47a was serially diluted from a maximum concentration of 50 µM (RIPK1) or 100 µM (RIPK3), and its ability to inhibit human RIPK1 or RIPK3 kinase activity was assessed using the ADP-Glo Kinase Assay (Promega). The reaction was allowed to proceed for 4 h before readouts were performed, and data were normalised to DMSO (0% inhibition) and either 1 µM GSK-481 (100% inhibition of RIPK1) or 1 µM GSK-872 (100% inhibition of RIPK3). Data are a summary of two independent experiments performed in duplicate, and the IC 50 and associated error (95% confidence interval) were determined using non-linear regression, as above. Each colour on the graph represents one independent experiment.

    Article Snippet: The following primary antibodies were used in this study: rat anti-MLKL clone 3H1 (available as MABC604 from Merck Millipore; 1:1000) was produced in-house as previously described [ ]; rabbit anti-human MLKL pS358 (ab187091, Abcam, Cambridge, United Kingdom; 1:4000); rabbit anti-mouse MLKL pS345 (ab196436, Abcam; 1:1000); rat anti-human RIPK3 (clone 1H2; available as MABC1640 from Merck Millipore; 1:1000) and rat anti-mouse RIPK3 (clone 8G7; available as MABC1595 from Merck Millipore; 1:1000) produced in-house as previously described [ , ]; rabbit anti-RIPK3 (2283, ProSci; 1:1000); rabbit anti-human RIPK3 pS227 (ab209384, Abcam; 1:2000); rabbit anti-mouse RIPK3 pT231/pS232 (GEN135-35-9, kindly supplied by Genentech, South San Francisco, CA, USA [ ]; 1:2000); mouse anti-RIPK1 (610458, BD Transduction Laboratories, San Jose, CA, USA; 1:1000); rabbit anti-RIPK1 (3493, Cell Signalling Technology, Danvers, MA, USA; 1:1000); rabbit anti-human RIPK1 pS166 (65746, Cell Signalling Technology; 1:1000); rabbit anti-mouse RIPK1 pS166 (31122, Cell Signalling Technology; 1:1000); rabbit anti-mouse CASP8 (4927, Cell Signalling Technology; 1:1000); rabbit anti-BAX (2772, Cell Signalling Technology; 1:1000); rabbit anti-BAX (06-536, Upstate Biotechnology, Lake Placid, NY, USA; 1:1000); rabbit anti-GAPDH (2118, Cell Signalling Technology; 1:1000); and mouse anti-β-Actin (A1978, Sigma-Aldrich; 1:3000).

    Techniques: Western Blot, Binding Assay, Concentration Assay, Activity Assay, Kinase Assay, Inhibition

    A The cell death induced by forced dimerisation of the human MLKL NTD involves two distinct processes. In the first, MLKL can self-associate into discrete oligomers on addition of doxycycline. In the second, addition of coumermycin promotes the assembly of large multimeric complexes. AMG-47a is unable to inhibit the second process, thus we propose it prevents membrane attachment, translocation, or oligomerisation of the human MLKL NTD through its activity as a kinase inhibitor. As AMG-47a binds RIPK1, and as RIPK1 is required for this form of cell death, we propose the primary target of AMG-47a is RIPK1 in this context. B When a cell receives a necroptotic stimulus, a number of key events occur, beginning with assembly of a necrosome containing RIPK1, RIPK3, and MLKL. This triggers a number of events, beginning with the phosphorylation of MLKL by RIPK3, which enables a conformational change in MLKL that allows oligomerisation, translocation to cellular membranes, and ultimately cell death. Our data suggest an additional role for RIPK1 in human necroptosis by supporting the conformational change, oligomerisation, or membrane translocation of MLKL following phosphorylation by RIPK3.

    Journal: Cell Death & Disease

    Article Title: The Lck inhibitor, AMG-47a, blocks necroptosis and implicates RIPK1 in signalling downstream of MLKL

    doi: 10.1038/s41419-022-04740-w

    Figure Lengend Snippet: A The cell death induced by forced dimerisation of the human MLKL NTD involves two distinct processes. In the first, MLKL can self-associate into discrete oligomers on addition of doxycycline. In the second, addition of coumermycin promotes the assembly of large multimeric complexes. AMG-47a is unable to inhibit the second process, thus we propose it prevents membrane attachment, translocation, or oligomerisation of the human MLKL NTD through its activity as a kinase inhibitor. As AMG-47a binds RIPK1, and as RIPK1 is required for this form of cell death, we propose the primary target of AMG-47a is RIPK1 in this context. B When a cell receives a necroptotic stimulus, a number of key events occur, beginning with assembly of a necrosome containing RIPK1, RIPK3, and MLKL. This triggers a number of events, beginning with the phosphorylation of MLKL by RIPK3, which enables a conformational change in MLKL that allows oligomerisation, translocation to cellular membranes, and ultimately cell death. Our data suggest an additional role for RIPK1 in human necroptosis by supporting the conformational change, oligomerisation, or membrane translocation of MLKL following phosphorylation by RIPK3.

    Article Snippet: The following primary antibodies were used in this study: rat anti-MLKL clone 3H1 (available as MABC604 from Merck Millipore; 1:1000) was produced in-house as previously described [ ]; rabbit anti-human MLKL pS358 (ab187091, Abcam, Cambridge, United Kingdom; 1:4000); rabbit anti-mouse MLKL pS345 (ab196436, Abcam; 1:1000); rat anti-human RIPK3 (clone 1H2; available as MABC1640 from Merck Millipore; 1:1000) and rat anti-mouse RIPK3 (clone 8G7; available as MABC1595 from Merck Millipore; 1:1000) produced in-house as previously described [ , ]; rabbit anti-RIPK3 (2283, ProSci; 1:1000); rabbit anti-human RIPK3 pS227 (ab209384, Abcam; 1:2000); rabbit anti-mouse RIPK3 pT231/pS232 (GEN135-35-9, kindly supplied by Genentech, South San Francisco, CA, USA [ ]; 1:2000); mouse anti-RIPK1 (610458, BD Transduction Laboratories, San Jose, CA, USA; 1:1000); rabbit anti-RIPK1 (3493, Cell Signalling Technology, Danvers, MA, USA; 1:1000); rabbit anti-human RIPK1 pS166 (65746, Cell Signalling Technology; 1:1000); rabbit anti-mouse RIPK1 pS166 (31122, Cell Signalling Technology; 1:1000); rabbit anti-mouse CASP8 (4927, Cell Signalling Technology; 1:1000); rabbit anti-BAX (2772, Cell Signalling Technology; 1:1000); rabbit anti-BAX (06-536, Upstate Biotechnology, Lake Placid, NY, USA; 1:1000); rabbit anti-GAPDH (2118, Cell Signalling Technology; 1:1000); and mouse anti-β-Actin (A1978, Sigma-Aldrich; 1:3000).

    Techniques: Translocation Assay, Activity Assay

    (A) IAV-induced cell death kinetics in primary wild-type (WT), Zbp1−/−, and Ripk3−/− MEFs.

    Journal: Cell

    Article Title: Influenza Virus Z-RNAs Induce ZBP1-Mediated Necroptosis

    doi: 10.1016/j.cell.2020.02.050

    Figure Lengend Snippet: (A) IAV-induced cell death kinetics in primary wild-type (WT), Zbp1−/−, and Ripk3−/− MEFs.

    Article Snippet: Rabbit polyclonal anti-RIPK3 , ProSci , Cat#2283; RRID: AB_203256.

    Techniques:

    (A) IAV-infected (PR8, MOI = 2) WT MEFs were lysed at the indicated times p.i., separated into nuclear and cytoplasmic fractions, and examined for ZBP1, MLKL, RIPK3, and viral proteins by immunoblotting. Immunoblotting for GAPDH and histone H3 was used to confirm purity of cytoplasmic and nuclear fractions.

    Journal: Cell

    Article Title: Influenza Virus Z-RNAs Induce ZBP1-Mediated Necroptosis

    doi: 10.1016/j.cell.2020.02.050

    Figure Lengend Snippet: (A) IAV-infected (PR8, MOI = 2) WT MEFs were lysed at the indicated times p.i., separated into nuclear and cytoplasmic fractions, and examined for ZBP1, MLKL, RIPK3, and viral proteins by immunoblotting. Immunoblotting for GAPDH and histone H3 was used to confirm purity of cytoplasmic and nuclear fractions.

    Article Snippet: Rabbit polyclonal anti-RIPK3 , ProSci , Cat#2283; RRID: AB_203256.

    Techniques: Infection, Western Blot

    (A) Survival analysis of age- and sex-matched WT (C57BL/6J) (n = 9), Zbp1−/− (n = 9), Ripk3−/− (n = 9), and Mlkl−/− (n = 8) mice infected with a modestly lethal (~LD20) dose of IAV (PR8, 2,500 EID50/mouse intranasally [i.n.]).

    Journal: Cell

    Article Title: Influenza Virus Z-RNAs Induce ZBP1-Mediated Necroptosis

    doi: 10.1016/j.cell.2020.02.050

    Figure Lengend Snippet: (A) Survival analysis of age- and sex-matched WT (C57BL/6J) (n = 9), Zbp1−/− (n = 9), Ripk3−/− (n = 9), and Mlkl−/− (n = 8) mice infected with a modestly lethal (~LD20) dose of IAV (PR8, 2,500 EID50/mouse intranasally [i.n.]).

    Article Snippet: Rabbit polyclonal anti-RIPK3 , ProSci , Cat#2283; RRID: AB_203256.

    Techniques: Infection

    KEY RESOURCES TABLE

    Journal: Cell

    Article Title: Influenza Virus Z-RNAs Induce ZBP1-Mediated Necroptosis

    doi: 10.1016/j.cell.2020.02.050

    Figure Lengend Snippet: KEY RESOURCES TABLE

    Article Snippet: Rabbit polyclonal anti-RIPK3 , ProSci , Cat#2283; RRID: AB_203256.

    Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, SYBR Green Assay, Immunoprecipitation, RNA Sequencing Assay, Plasmid Preparation, Software