Journal: Science Immunology

Article Title: ZBP1-dependent inflammatory cell death, PANoptosis, and cytokine storm disrupt IFN therapeutic efficacy during coronavirus infection

doi: 10.1126/sciimmunol.abo6294

Figure Lengend Snippet: ( A – C ) Immunoblot analysis of ( A ) pro- (P53) and activated (P30) gasdermin D (GSDMD), pro- (P53) and activated (P34) gasdermin E (GSDME); ( B ) pro- (P55) and cleaved caspase-8 (CASP8; P18), pro- (P35) and cleaved caspase-3 (CASP3; P19 and P17) and pro- (P35) and cleaved caspase-7 (CASP7; P20); and ( C ) phosphorylated MLKL (pMLKL), total MLKL (tMLKL), phosphorylated RIPK3 (pRIPK3) and total RIPK3 (tRIPK3) in the lung samples from mock- or IFN-β–treated wild type (WT) mice with or without mouse hepatitis virus (MHV) infection 3 days post-infection. ( D – I ) Immunoblot analysis of ( D , G ) pro- (P45) and activated (P20) caspase-1 (CASP1), pro- (P53) and activated (P30) GSDMD, pro- (P53) and activated (P34) GSDME; ( E , H ) pro- (P55) and cleaved CASP8 (P18), pro- (P35) and cleaved CASP3 (P19 and P17) and pro- (P35) and cleaved CASP7 (P20); ( F) pMLKL, tMLKL, pRIPK3 and tRIPK3; and ( I ) pRIPK3 and tRIPK3 in mock- or IFN-β–treated bone marrow-derived macrophages (BMDMs) or THP-1 cells during MHV or SARS-CoV-2 infection, respectively. Actin was used as the internal control. Molecular weight marker sizes in kDa are indicated in small font on the left of each blot. Asterisk denotes non-specific bands ( A, G ). Data are representative of at least three independent experiments.

Article Snippet: Following electrophoretic transfer of proteins onto PVDF membranes (Millipore, IPVH00010), non-specific binding was blocked by incubation with 5% skim milk; then membranes were incubated with the following primary antibodies: anti-caspase-1 (AdipoGen, AG-20B-0042, 1:1000), anti-caspase-3 (CST, #9662, 1:1000), anti-cleaved caspase-3 (CST, #9661, 1:1000), anti-caspase-7 (CST, #9492, 1:1000), anti-cleaved caspase-7 (CST, #9491, 1:1000), anti-caspase-8 (CST, #4927, 1:1000), anti-cleaved caspase-8 (CST, #8592, 1:1000), anti-pRIPK3 (CST, #91702S, 1:1000), anti-RIPK3 (ProSci, #2283, 1:1000), anti-pMLKL (CST, #37333, 1:1000), anti-MLKL (Abgent, AP14272b, 1:1000), anti-GSDMD (Abcam, ab209845, 1:1000), anti-GSDME (Abcam, ab215191, 1:1000), anti-pSTAT1 (CST, #7649, 1:1000), anti-STAT1 (CST, #14994, 1:1000), anti-ZBP1 (AdipoGen, AG-20B-0010, 1:1000), anti-β-actin (Proteintech, 66009-1-IG, 1:5000), human anti-caspase-1 (R&D systems; Cat# MAB6215, 1:1000), human anti-caspase-8 (Enzo, ALX-804-242, 1:1000), human anti-GSDMD (Abcam, ab210070, 1:1000), human anti-tMLKL (Abcam, ab184718, 1:1000), human anti-β-actin (CST, 4970, 1:1000), human anti-ZBP1 (R&D systems; Cat# AF6309, 1:1000) and anti-ISG15 (Santa Cruz, sc-166755).

Techniques: Western Blot, Infection, Derivative Assay, Molecular Weight, Marker

Journal: Science Immunology

Article Title: ZBP1-dependent inflammatory cell death, PANoptosis, and cytokine storm disrupt IFN therapeutic efficacy during coronavirus infection

doi: 10.1126/sciimmunol.abo6294

Figure Lengend Snippet: ( A – C ) Immunoblot analysis of ( A ) pro- (P53) and activated (P30) gasdermin D (GSDMD), pro- (P53) and activated (P34) gasdermin E (GSDME); ( B ) pro- (P55) and cleaved caspase-8 (CASP8; P18), pro- (P35) and cleaved caspase-3 (CASP3; P19 and P17) and pro- (P35) and cleaved caspase-7 (CASP7; P20); and ( C ) phosphorylated MLKL (pMLKL), total MLKL (tMLKL), phosphorylated RIPK3 (pRIPK3) and total RIPK3 (tRIPK3) in the lung samples from PBS-treated mice or mouse hepatitis virus (MHV)-infected wild type (WT) and Zbp1 –/– mice treated with IFN-β harvested 3 days after infection. ( D – F ) Immunoblot analysis of ( D ) pro- (P45) and activated (P20) caspase-1 (CASP1), pro- (P53) and activated (P30) GSDMD, pro- (P53) and activated (P34) GSDME; ( E ) pro- (P55) and cleaved (P18) CASP8, pro- (P35) and cleaved (P19 and P17) CASP3 and pro- (P35) and cleaved (P20) CASP7; and ( F ) pMLKL, tMLKL and ZBP1 in PBS- or IFN-β–treated WT, Zbp1 –/– and Zbp1 ∆Za2 bone marrow-derived macrophages (BMDMs) during MHV infection. Actin was used as the internal control. Data are representative of at least three independent experiments.

Article Snippet: Following electrophoretic transfer of proteins onto PVDF membranes (Millipore, IPVH00010), non-specific binding was blocked by incubation with 5% skim milk; then membranes were incubated with the following primary antibodies: anti-caspase-1 (AdipoGen, AG-20B-0042, 1:1000), anti-caspase-3 (CST, #9662, 1:1000), anti-cleaved caspase-3 (CST, #9661, 1:1000), anti-caspase-7 (CST, #9492, 1:1000), anti-cleaved caspase-7 (CST, #9491, 1:1000), anti-caspase-8 (CST, #4927, 1:1000), anti-cleaved caspase-8 (CST, #8592, 1:1000), anti-pRIPK3 (CST, #91702S, 1:1000), anti-RIPK3 (ProSci, #2283, 1:1000), anti-pMLKL (CST, #37333, 1:1000), anti-MLKL (Abgent, AP14272b, 1:1000), anti-GSDMD (Abcam, ab209845, 1:1000), anti-GSDME (Abcam, ab215191, 1:1000), anti-pSTAT1 (CST, #7649, 1:1000), anti-STAT1 (CST, #14994, 1:1000), anti-ZBP1 (AdipoGen, AG-20B-0010, 1:1000), anti-β-actin (Proteintech, 66009-1-IG, 1:5000), human anti-caspase-1 (R&D systems; Cat# MAB6215, 1:1000), human anti-caspase-8 (Enzo, ALX-804-242, 1:1000), human anti-GSDMD (Abcam, ab210070, 1:1000), human anti-tMLKL (Abcam, ab184718, 1:1000), human anti-β-actin (CST, 4970, 1:1000), human anti-ZBP1 (R&D systems; Cat# AF6309, 1:1000) and anti-ISG15 (Santa Cruz, sc-166755).

Techniques: Western Blot, Infection, Derivative Assay

Journal: Biochemical Journal

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

doi: 10.1042/BCJ20230035

Figure Lengend 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.

Article Snippet: Mouse RIPK3 (CETSA) , Rabbit anti-RIPK3 , ProSci , cat #2283 , 1 : 1000.

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

Journal: Biochemical Journal

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

doi: 10.1042/BCJ20230035

Figure Lengend 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.

Article Snippet: Mouse RIPK3 (CETSA) , Rabbit anti-RIPK3 , ProSci , cat #2283 , 1 : 1000.

Techniques: Staining, Flow Cytometry, Live Cell Imaging

Journal: Biochemical Journal

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

doi: 10.1042/BCJ20230035

Figure Lengend 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.

Article Snippet: Mouse RIPK3 (CETSA) , Rabbit anti-RIPK3 , ProSci , cat #2283 , 1 : 1000.

Techniques: Binding Assay, Expressing, Western Blot

Journal: Biochemical Journal

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

doi: 10.1042/BCJ20230035

Figure Lengend 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.

Article Snippet: Mouse RIPK3 (CETSA) , Rabbit anti-RIPK3 , ProSci , cat #2283 , 1 : 1000.

Techniques:

Journal: Biochemical Journal

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

doi: 10.1042/BCJ20230035

Figure Lengend 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.

Article Snippet: Mouse RIPK3 (CETSA) , Rabbit anti-RIPK3 , ProSci , cat #2283 , 1 : 1000.

Techniques: In Vitro, Western Blot

Journal: Biochemical Journal

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

doi: 10.1042/BCJ20230035

Figure Lengend Snippet:

Article Snippet: Mouse RIPK3 (CETSA) , Rabbit anti-RIPK3 , ProSci , cat #2283 , 1 : 1000.

Techniques: Western Blot, Produced, Transduction

Journal: Biochemical Journal

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

doi: 10.1042/BCJ20230035

Figure Lengend Snippet:

Article Snippet: Mouse RIPK3 (CETSA) , Rabbit anti-RIPK3 , ProSci , cat #2283 , 1 : 1000.

Techniques:

Journal: Biochemical Journal

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

doi: 10.1042/BCJ20230035

Figure Lengend 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.

Article Snippet: Mouse RIPK3 (CETSA) , Rabbit anti-RIPK3 , ProSci , cat #2283 , 1 : 1000.

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

Journal: Biochemical Journal

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

doi: 10.1042/BCJ20230035

Figure Lengend 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.

Article Snippet: Mouse RIPK3 (CETSA) , Rabbit anti-RIPK3 , ProSci , cat #2283 , 1 : 1000.

Techniques: Staining, Flow Cytometry, Live Cell Imaging

Journal: Biochemical Journal

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

doi: 10.1042/BCJ20230035

Figure Lengend 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.

Article Snippet: Mouse RIPK3 (CETSA) , Rabbit anti-RIPK3 , ProSci , cat #2283 , 1 : 1000.

Techniques: Binding Assay, Expressing, Western Blot

Journal: Biochemical Journal

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

doi: 10.1042/BCJ20230035

Figure Lengend 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.

Article Snippet: Mouse RIPK3 (CETSA) , Rabbit anti-RIPK3 , ProSci , cat #2283 , 1 : 1000.

Techniques:

Journal: Biochemical Journal

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

doi: 10.1042/BCJ20230035

Figure Lengend 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.

Article Snippet: Mouse RIPK3 (CETSA) , Rabbit anti-RIPK3 , ProSci , cat #2283 , 1 : 1000.

Techniques: In Vitro, Western Blot

Journal: Biochemical Journal

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

doi: 10.1042/BCJ20230035

Figure Lengend Snippet:

Article Snippet: Mouse RIPK3 (CETSA) , Rabbit anti-RIPK3 , ProSci , cat #2283 , 1 : 1000.

Techniques: Western Blot, Produced, Transduction

Journal: Biochemical Journal

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

doi: 10.1042/BCJ20230035

Figure Lengend Snippet:

Article Snippet: Mouse RIPK3 (CETSA) , Rabbit anti-RIPK3 , ProSci , cat #2283 , 1 : 1000.

Techniques:

Journal: Life Science Alliance

Article Title: Integrated stress response restricts macrophage necroptosis

doi: 10.26508/lsa.202101260

Figure Lengend Snippet: Necroptosis signaling was analyzed by immunoblotting at the indicated time after the induction of necroptosis by TNF treatment. (A, B, C, D, E) Lysates from primary BMDMs pre-stressed with (A) thapsigargin (Thaps), (B) brefeldin A, (C) tunicamycin (Tunica), (D) MG132, or (E) Thaps and treated with zVAD + TNF were collected at the indicated time-points. Necroptosis signaling was examined by immunostaining for phosphorylation of MLKL and RIPK3 (p-MLKL and p-RIPK3) and phosphorylation of RIPK1 (p-RIPK1) at serine-166 (S166) or serine-321 (S321) and activation of the integrated stress response by phosphorylation of eIF2α (p-eIF2α). Immunoblots are representative of at least two independent experiments.

Article Snippet: Primary antibodies against phospho-MLKL (Ser345) (37333; Cell Signaling Technologies [CST], 1:1,000), MLKL (37705; CST, 1:1,000), phospho-RIPK3 (Thr231/Ser232) (91702; CST, 1:1,000), RIPK3 (2283; ProSci, 1:1,000), phospho-eIF2α (Ser51) (3398; CST, 1:1,000), eIF2α (9722; CST, 1:1,000), RIPK1 (3493; CST, 1:1,000), phospho-RIPK1 (Ser166) (53286; CST, 1:1,000), phospho-RIPK1 (Ser321) (38662; CST, 1:1,000), DDX3X (A300-474A; Bethyl Laboratories, 1:1,000), PERK (3192; CST, 1:1,000), or β-actin (8457; CST, 1:1,000) were incubated overnight at 4°C followed by appropriate secondary antibodies conjugated with HRP incubated for 1 h at room temperature (Jackson ImmunoResearch).

Techniques: Western Blot, Immunostaining, Activation Assay

Journal: Life Science Alliance

Article Title: Integrated stress response restricts macrophage necroptosis

doi: 10.26508/lsa.202101260

Figure Lengend Snippet: Primary BMDMs were stimulated as indicated. (A, B, C, D) Quantification of cell death from automated image analysis of Sytox Green–positive nuclei at indicated time-points in (A) WT BMDMs, (B) Ripk1 KD/KD BMDMs, (C) Ripk3 −/− BMDMs, or (D) Mlkl −/− BMDMs. (E) WT BMDM cell death was similarly quantified at indicated time-points in pre-stressed (thapsigargin [Thaps]-treated) cells subsequently treated with cycloheximide (CHX). Significance was determined by two-way ANOVA followed by Tukey’s multiple comparisons test, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. Data are generated from three images per replicate well (n = 3) and are representative of at least three independent biological replicate experiments. Data are presented as mean ± SEM.

Article Snippet: Primary antibodies against phospho-MLKL (Ser345) (37333; Cell Signaling Technologies [CST], 1:1,000), MLKL (37705; CST, 1:1,000), phospho-RIPK3 (Thr231/Ser232) (91702; CST, 1:1,000), RIPK3 (2283; ProSci, 1:1,000), phospho-eIF2α (Ser51) (3398; CST, 1:1,000), eIF2α (9722; CST, 1:1,000), RIPK1 (3493; CST, 1:1,000), phospho-RIPK1 (Ser166) (53286; CST, 1:1,000), phospho-RIPK1 (Ser321) (38662; CST, 1:1,000), DDX3X (A300-474A; Bethyl Laboratories, 1:1,000), PERK (3192; CST, 1:1,000), or β-actin (8457; CST, 1:1,000) were incubated overnight at 4°C followed by appropriate secondary antibodies conjugated with HRP incubated for 1 h at room temperature (Jackson ImmunoResearch).

Techniques: Generated