anti mouse ripk3  (ProSci Incorporated)


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    ProSci Incorporated anti mouse ripk3
    Anti Mouse 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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    mouse ripk3 cetsa  (ProSci Incorporated)


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    ProSci Incorporated mouse ripk3 cetsa
    ( 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.
    Mouse Ripk3 Cetsa, 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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    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:

    anti mouse ripk3  (ProSci Incorporated)


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    ProSci Incorporated anti mouse ripk3
    Anti Mouse 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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    anti mouse ripk3  (ProSci Incorporated)


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    ProSci Incorporated anti mouse ripk3
    TNF treatment induces activation of AMPK. ( A ) L929 cells were transfected with non-targeting (siCtrl) or <t>Ripk3</t> siRNAs (si Ripk3 ). 48 h post transfection, cells were exposed to 10 ng/ml TNF and 30 µM QVD (TQ) for the indicated times. Then, whole cell lysates were subjected to SDS-PAGE and immunoblotting for indicated proteins. A compilation of representative immunoblots is shown; three ACTB immunblots are shown, but each protein was normalized to its corresponding loading control. The density of each protein band was divided by the average of the density of all bands from the same protein on the membrane. Fold changes were calculated by dividing each normalized ratio (protein to loading control) by the average of the ratios of the control lane (scr, 0 h TQ). Results are mean + SD from at least 3 independent experiments. Statistical analysis was done by repeated measures two-way ANOVA (corrected by Sidak’s multiple comparisons test between siRNAs and corrected by Tukey’s multiple comparisons test between time points). Statistically significant differences within non-targeting siRNA-transfected cells (compared to scr, 0 h TQ) are depicted as letters directly above the bars. * or a: P < 0.05, ** or b: P < 0.01, *** or c: P < 0.001, **** or d: P < 0.0001. ( B ) Ripk3 WT and KO MEFs were exposed to indicated treatments (medium [M], 30 ng/ml TNF [T], 100 nM SMAC-mimetic [S], 20 µM z-VAD [Z]) for indicated times. Then, cells were lysed and cleared cellular lysates were subjected to SDS-PAGE and analyzed by immunoblotting for indicated proteins
    Anti Mouse 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
    https://www.bioz.com/result/anti mouse ripk3/product/ProSci Incorporated
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    1) Product Images from "TNF-induced necroptosis initiates early autophagy events via RIPK3-dependent AMPK activation, but inhibits late autophagy"

    Article Title: TNF-induced necroptosis initiates early autophagy events via RIPK3-dependent AMPK activation, but inhibits late autophagy

    Journal: Autophagy

    doi: 10.1080/15548627.2021.1899667

    TNF treatment induces activation of AMPK. ( A ) L929 cells were transfected with non-targeting (siCtrl) or Ripk3 siRNAs (si Ripk3 ). 48 h post transfection, cells were exposed to 10 ng/ml TNF and 30 µM QVD (TQ) for the indicated times. Then, whole cell lysates were subjected to SDS-PAGE and immunoblotting for indicated proteins. A compilation of representative immunoblots is shown; three ACTB immunblots are shown, but each protein was normalized to its corresponding loading control. The density of each protein band was divided by the average of the density of all bands from the same protein on the membrane. Fold changes were calculated by dividing each normalized ratio (protein to loading control) by the average of the ratios of the control lane (scr, 0 h TQ). Results are mean + SD from at least 3 independent experiments. Statistical analysis was done by repeated measures two-way ANOVA (corrected by Sidak’s multiple comparisons test between siRNAs and corrected by Tukey’s multiple comparisons test between time points). Statistically significant differences within non-targeting siRNA-transfected cells (compared to scr, 0 h TQ) are depicted as letters directly above the bars. * or a: P < 0.05, ** or b: P < 0.01, *** or c: P < 0.001, **** or d: P < 0.0001. ( B ) Ripk3 WT and KO MEFs were exposed to indicated treatments (medium [M], 30 ng/ml TNF [T], 100 nM SMAC-mimetic [S], 20 µM z-VAD [Z]) for indicated times. Then, cells were lysed and cleared cellular lysates were subjected to SDS-PAGE and analyzed by immunoblotting for indicated proteins
    Figure Legend Snippet: TNF treatment induces activation of AMPK. ( A ) L929 cells were transfected with non-targeting (siCtrl) or Ripk3 siRNAs (si Ripk3 ). 48 h post transfection, cells were exposed to 10 ng/ml TNF and 30 µM QVD (TQ) for the indicated times. Then, whole cell lysates were subjected to SDS-PAGE and immunoblotting for indicated proteins. A compilation of representative immunoblots is shown; three ACTB immunblots are shown, but each protein was normalized to its corresponding loading control. The density of each protein band was divided by the average of the density of all bands from the same protein on the membrane. Fold changes were calculated by dividing each normalized ratio (protein to loading control) by the average of the ratios of the control lane (scr, 0 h TQ). Results are mean + SD from at least 3 independent experiments. Statistical analysis was done by repeated measures two-way ANOVA (corrected by Sidak’s multiple comparisons test between siRNAs and corrected by Tukey’s multiple comparisons test between time points). Statistically significant differences within non-targeting siRNA-transfected cells (compared to scr, 0 h TQ) are depicted as letters directly above the bars. * or a: P < 0.05, ** or b: P < 0.01, *** or c: P < 0.001, **** or d: P < 0.0001. ( B ) Ripk3 WT and KO MEFs were exposed to indicated treatments (medium [M], 30 ng/ml TNF [T], 100 nM SMAC-mimetic [S], 20 µM z-VAD [Z]) for indicated times. Then, cells were lysed and cleared cellular lysates were subjected to SDS-PAGE and analyzed by immunoblotting for indicated proteins

    Techniques Used: Activation Assay, Transfection, SDS Page, Western Blot

    TNF treatment induces ATG14 and ATG16L1 puncta formation via RIPK3 and AMPK. ( A and B ) WT and ripk3 KO L929 cells were exposed to indicated treatments (medium [M], 10 ng/ml TNF [T], 30 µM QVD [Q], 5 µM GSK’872 [G]) for 3 h. After that, cells were fixed and subjected to ATG14 (A) or ATG16L1 (B) immunostaining using anti-ATG14 (Santa Cruz Biotechnology, sc-164767) or anti-ATG16L1 antibodies (MBL International, PM040) and IRDye® 680RD donkey anti-goat or Alexa Fluor®488-conjugated goat anti-rabbit IgG (H + L) secondary antibodies. Puncta quantification was done using ImageJ software. Data represent mean + SD. A minimum of 120 (A) or 261 cells (B) was analyzed. ( C ) WT L929 cells were transfected with non-targeting (siCtrl) or Prkaa1/Prkaa2 siRNAs (si Prkaa1/ si Prkaa2 ). 48 h post transfection, cells were left untreated (medium, M) or exposed to 10 ng/ml TNF and 30 µM QVD (TQ) for 3 h. Then, cells were fixed and subjected to ATG16L1 immunostaining using anti-ATG16L1 antibodies (MBL International, PM040) and Alexa Fluor®488-conjugated goat anti-rabbit IgG (H + L) secondary antibodies. Puncta quantification was done using ImageJ software. Data represent mean + SD. A minimum of 655 cells was analyzed. ( D ) WT L929 cells were left untreated (medium, M) or exposed to indicated treatments (10 ng/ml TNF [T], 30 µM QVD [Q], 5 µM AMPK inhibitor dorsomorphin) for 3 h. Then, cells were fixed and subjected to ATG16L1 immunostaining using anti-ATG16L1 antibodies (MBL International, PM040) and Alexa Fluor®488-conjugated goat anti-rabbit IgG (H + L) secondary antibodies. Puncta quantification was done using ImageJ software. Data represent mean + SD. A minimum of 122 cells was analyzed. ( A-D ) Statistical analysis was performed using ordinary one-way ANOVA (corrected by Tukey’s multiple comparisons test) for A, B and D; or two-way ANOVA (corrected by Tukey’s multiple comparisons test) for C. For B, statistical analysis was additionally performed using unpaired t test with Welch’s correction (TQ treatment of WT vs. ripk3 KO cells). **** P < 0.0001. Scale bar: 20 µm
    Figure Legend Snippet: TNF treatment induces ATG14 and ATG16L1 puncta formation via RIPK3 and AMPK. ( A and B ) WT and ripk3 KO L929 cells were exposed to indicated treatments (medium [M], 10 ng/ml TNF [T], 30 µM QVD [Q], 5 µM GSK’872 [G]) for 3 h. After that, cells were fixed and subjected to ATG14 (A) or ATG16L1 (B) immunostaining using anti-ATG14 (Santa Cruz Biotechnology, sc-164767) or anti-ATG16L1 antibodies (MBL International, PM040) and IRDye® 680RD donkey anti-goat or Alexa Fluor®488-conjugated goat anti-rabbit IgG (H + L) secondary antibodies. Puncta quantification was done using ImageJ software. Data represent mean + SD. A minimum of 120 (A) or 261 cells (B) was analyzed. ( C ) WT L929 cells were transfected with non-targeting (siCtrl) or Prkaa1/Prkaa2 siRNAs (si Prkaa1/ si Prkaa2 ). 48 h post transfection, cells were left untreated (medium, M) or exposed to 10 ng/ml TNF and 30 µM QVD (TQ) for 3 h. Then, cells were fixed and subjected to ATG16L1 immunostaining using anti-ATG16L1 antibodies (MBL International, PM040) and Alexa Fluor®488-conjugated goat anti-rabbit IgG (H + L) secondary antibodies. Puncta quantification was done using ImageJ software. Data represent mean + SD. A minimum of 655 cells was analyzed. ( D ) WT L929 cells were left untreated (medium, M) or exposed to indicated treatments (10 ng/ml TNF [T], 30 µM QVD [Q], 5 µM AMPK inhibitor dorsomorphin) for 3 h. Then, cells were fixed and subjected to ATG16L1 immunostaining using anti-ATG16L1 antibodies (MBL International, PM040) and Alexa Fluor®488-conjugated goat anti-rabbit IgG (H + L) secondary antibodies. Puncta quantification was done using ImageJ software. Data represent mean + SD. A minimum of 122 cells was analyzed. ( A-D ) Statistical analysis was performed using ordinary one-way ANOVA (corrected by Tukey’s multiple comparisons test) for A, B and D; or two-way ANOVA (corrected by Tukey’s multiple comparisons test) for C. For B, statistical analysis was additionally performed using unpaired t test with Welch’s correction (TQ treatment of WT vs. ripk3 KO cells). **** P < 0.0001. Scale bar: 20 µm

    Techniques Used: Immunostaining, Software, Transfection

    RIPK3 interacts with AMPK. ( A ) S100 extracts of MEFs were separated by size-exclusion chromatography on a Superose 6 increase column. Fractions were analyzed by immunoblotting for the indicated proteins. The diagram shows protein levels for fractions 16–40 and the density of each protein band was divided by the average of the density of all bands from the same protein on the membrane. ( B ) HEK293 cells were left untransfected or were transfected with a vector encoding 3xFLAG-HsRIPK3 for 24 h. Then, cells were lysed and cleared cellular lysates were subjected to immunopurification using anti-FLAG beads. Purified proteins were subjected to SDS-PAGE and analyzed by immunoblotting for FLAG and AMPK. ( C ) HEK293 cells were left untransfected or were transfected with a vector encoding 3xFLAG-HsRIPK3 for 24 h. Then, cells were lysed and cleared cellular lysates were subjected to immunopurification using anti-AMPK antibodies. Purified proteins were subjected to SDS-PAGE and analyzed by immunoblotting for FLAG and AMPK. ( D ) L929 cells were lysed and cleared cellular lysates were subjected to immunopurification using anti-IgG or anti-RIPK3 antibodies. Purified proteins were subjected to SDS-PAGE and analyzed by immunoblotting for AMPK and RIPK3. ( E ) GST or GST-MmRIPK3 immobilized on glutathione-Sepharose beads was incubated with His-AMPK [His-HsPRKAA1 (11–559) + HsPRKAB2 (1–272) + HsPRKAG1 (1–331)] overnight. After washing the beads, bound proteins were eluted by boiling for 10 min at 95°C. Proteins were subjected to SDS-PAGE and analyzed by immunoblotting for AMPK and GST. ( F ) ripk3 KO MEFs were retrovirally transfected with empty vector or cDNA encoding 3xFLAG-MmRIPK3. Cells were seeded onto glass coverslips. The next day, cells were fixed and analyzed using proximity ligation assay as described in the material and methods section (anti-RIPK3: Prosci, 2283; anti-PRKAA1/2: Cell Signaling Technology, 2793). Nuclei were stained with DAPI. Signals and nuclei per image were counted and the signal:nuclei ratio was calculated. Data represent mean + SD. A minimum of 216 cells was analyzed. Statistical analysis was performed using an unpaired t test with Welch’s correction. **** P < 0.0001. Scale bar: 20 µm
    Figure Legend Snippet: RIPK3 interacts with AMPK. ( A ) S100 extracts of MEFs were separated by size-exclusion chromatography on a Superose 6 increase column. Fractions were analyzed by immunoblotting for the indicated proteins. The diagram shows protein levels for fractions 16–40 and the density of each protein band was divided by the average of the density of all bands from the same protein on the membrane. ( B ) HEK293 cells were left untransfected or were transfected with a vector encoding 3xFLAG-HsRIPK3 for 24 h. Then, cells were lysed and cleared cellular lysates were subjected to immunopurification using anti-FLAG beads. Purified proteins were subjected to SDS-PAGE and analyzed by immunoblotting for FLAG and AMPK. ( C ) HEK293 cells were left untransfected or were transfected with a vector encoding 3xFLAG-HsRIPK3 for 24 h. Then, cells were lysed and cleared cellular lysates were subjected to immunopurification using anti-AMPK antibodies. Purified proteins were subjected to SDS-PAGE and analyzed by immunoblotting for FLAG and AMPK. ( D ) L929 cells were lysed and cleared cellular lysates were subjected to immunopurification using anti-IgG or anti-RIPK3 antibodies. Purified proteins were subjected to SDS-PAGE and analyzed by immunoblotting for AMPK and RIPK3. ( E ) GST or GST-MmRIPK3 immobilized on glutathione-Sepharose beads was incubated with His-AMPK [His-HsPRKAA1 (11–559) + HsPRKAB2 (1–272) + HsPRKAG1 (1–331)] overnight. After washing the beads, bound proteins were eluted by boiling for 10 min at 95°C. Proteins were subjected to SDS-PAGE and analyzed by immunoblotting for AMPK and GST. ( F ) ripk3 KO MEFs were retrovirally transfected with empty vector or cDNA encoding 3xFLAG-MmRIPK3. Cells were seeded onto glass coverslips. The next day, cells were fixed and analyzed using proximity ligation assay as described in the material and methods section (anti-RIPK3: Prosci, 2283; anti-PRKAA1/2: Cell Signaling Technology, 2793). Nuclei were stained with DAPI. Signals and nuclei per image were counted and the signal:nuclei ratio was calculated. Data represent mean + SD. A minimum of 216 cells was analyzed. Statistical analysis was performed using an unpaired t test with Welch’s correction. **** P < 0.0001. Scale bar: 20 µm

    Techniques Used: Size-exclusion Chromatography, Western Blot, Transfection, Plasmid Preparation, Immu-Puri, Purification, SDS Page, Incubation, Proximity Ligation Assay, Staining

    RIPK3 directly phosphorylates PRKAA1 at T183. ( A ) For in vitro kinase assay, purified GST, GST-HsPRKAA1(1–278) and GST-HsPRKAA1(279–559) were incubated with activated RIPK3 and [γ- 32 P]-ATP. The reactions were subjected to SDS-PAGE. After Coomassie Brilliant Blue staining and drying of the gels, autoradiography was performed. ( B ) GST-HsPRKAA1 WT and the T183A mutant were purified and were incubated with activated RIPK3 and cold ATP. The reactions were subjected to SDS-PAGE and analyzed by immunoblotting for phospho-PRKAA1/2 T183/T172 and AMPK. ( C ) GST-HsPRKAA1 WT and the T183A mutant were incubated with activated RIPK3 and cold ATP with or without alkaline phosphatase. The reactions were subjected to SDS-PAGE and analyzed by immunoblotting for phospho-PRKAA1/2 T183/T172 and AMPK. ( D ) GST-HsPRKAA1 WT was incubated with activated RIPK3 and cold ATP with or without 50 µM GSK’872. The reactions were subjected to SDS-PAGE and analyzed by immunoblotting for phospho-PRKAA1/2 T183/T172 and AMPK. ( E ) HEK293 cells were left untransfected or were transfected with cDNA encoding either 3xFLAG-HsRIPK3 WT or 3xFLAG-HsRIPK3 kinase-dead (KD) for 24 h. After that, cells were treated with 30 ng/ml TNF + 30 µM QVD for 24 h. Then, cells were lysed and cleared cellular lysates were subjected to SDS-PAGE and analyzed by immunoblotting for phospho-PRKAA1/2 T183/T172, AMPK, FLAG and ACTB. ( F ) ripk3 KO MEFs were retrovirally transfected with empty vector or cDNA encoding 3xFLAG-MmRIPK3. Cells were seeded onto glass coverslips. The next day, the cells were left untreated (medium, M) or treated with 30 ng/ml TNF + 100 nM SMAC-mimetic + 20 µM z-VAD (TSZ) for 3 h. Then cells were fixed and analyzed using proximity ligation assay as described in the material and methods section (anti-phospho-PRKAA1/2 T183/T172: Cell Signaling Technology, 2535; anti-PRKAA1/2: Cell Signaling Technology, 2793). Nuclei were stained with DAPI. Signals and nuclei per image were counted and the signal:nuclei ratio was calculated. Data represent mean + SD. A minimum of 107 cells was analyzed. Statistical analysis was performed using ordinary two-way ANOVA (corrected by Tukey’s multiple comparisons test). **** P < 0.0001. Scale bar: 20 µm. ( G ) L929 cells were transiently transfected with cDNA encoding either 3xFLAG-HsPRKAG1 WT or R299G for 24 h. After that, cells were treated with or without 10 ng/ml TNF + 30 µM QVD (TQ) for 2 h. Then, cells were lysed and cleared cellular lysates were subjected to immunopurification using anti-FLAG beads. Purified proteins were subjected to SDS-PAGE and analyzed by immunoblotting for indicated proteins
    Figure Legend Snippet: RIPK3 directly phosphorylates PRKAA1 at T183. ( A ) For in vitro kinase assay, purified GST, GST-HsPRKAA1(1–278) and GST-HsPRKAA1(279–559) were incubated with activated RIPK3 and [γ- 32 P]-ATP. The reactions were subjected to SDS-PAGE. After Coomassie Brilliant Blue staining and drying of the gels, autoradiography was performed. ( B ) GST-HsPRKAA1 WT and the T183A mutant were purified and were incubated with activated RIPK3 and cold ATP. The reactions were subjected to SDS-PAGE and analyzed by immunoblotting for phospho-PRKAA1/2 T183/T172 and AMPK. ( C ) GST-HsPRKAA1 WT and the T183A mutant were incubated with activated RIPK3 and cold ATP with or without alkaline phosphatase. The reactions were subjected to SDS-PAGE and analyzed by immunoblotting for phospho-PRKAA1/2 T183/T172 and AMPK. ( D ) GST-HsPRKAA1 WT was incubated with activated RIPK3 and cold ATP with or without 50 µM GSK’872. The reactions were subjected to SDS-PAGE and analyzed by immunoblotting for phospho-PRKAA1/2 T183/T172 and AMPK. ( E ) HEK293 cells were left untransfected or were transfected with cDNA encoding either 3xFLAG-HsRIPK3 WT or 3xFLAG-HsRIPK3 kinase-dead (KD) for 24 h. After that, cells were treated with 30 ng/ml TNF + 30 µM QVD for 24 h. Then, cells were lysed and cleared cellular lysates were subjected to SDS-PAGE and analyzed by immunoblotting for phospho-PRKAA1/2 T183/T172, AMPK, FLAG and ACTB. ( F ) ripk3 KO MEFs were retrovirally transfected with empty vector or cDNA encoding 3xFLAG-MmRIPK3. Cells were seeded onto glass coverslips. The next day, the cells were left untreated (medium, M) or treated with 30 ng/ml TNF + 100 nM SMAC-mimetic + 20 µM z-VAD (TSZ) for 3 h. Then cells were fixed and analyzed using proximity ligation assay as described in the material and methods section (anti-phospho-PRKAA1/2 T183/T172: Cell Signaling Technology, 2535; anti-PRKAA1/2: Cell Signaling Technology, 2793). Nuclei were stained with DAPI. Signals and nuclei per image were counted and the signal:nuclei ratio was calculated. Data represent mean + SD. A minimum of 107 cells was analyzed. Statistical analysis was performed using ordinary two-way ANOVA (corrected by Tukey’s multiple comparisons test). **** P < 0.0001. Scale bar: 20 µm. ( G ) L929 cells were transiently transfected with cDNA encoding either 3xFLAG-HsPRKAG1 WT or R299G for 24 h. After that, cells were treated with or without 10 ng/ml TNF + 30 µM QVD (TQ) for 2 h. Then, cells were lysed and cleared cellular lysates were subjected to immunopurification using anti-FLAG beads. Purified proteins were subjected to SDS-PAGE and analyzed by immunoblotting for indicated proteins

    Techniques Used: In Vitro, Kinase Assay, Purification, Incubation, SDS Page, Staining, Autoradiography, Mutagenesis, Western Blot, Transfection, Plasmid Preparation, Proximity Ligation Assay, Immu-Puri

    Necroptosis inhibits lysosomal LC3 degradation. ( A ) L929 cells were left untreated (medium, M) or exposed to 30 µM QVD (Q), 20 nM bafilomycin A 1 (B), 10 ng/ml TNF (T), 10 ng/ml TNF + 30 µM QVD with or without 20 nM bafilomycin A 1 (TQ or TQB) for indicated times. Then, cells were lysed and cleared cellular lysates were subjected to SDS-PAGE and immunoblotting for indicated proteins. The density of each protein band was divided by the average of the density of all bands from the same protein on the membrane. Fold changes were calculated by dividing each normalized ratio (protein to loading control) by the average of the ratios of the control lane (medium). Statistical graphics represents mean + SD (n = 4). ( B ) L929 WT, ripk3 KO or MLKL KO cells were exposed to 10 ng/ml TNF and 30 µM QVD (TQ) for indicated times. Then, cells were lysed and cleared cellular lysates were subjected to SDS-PAGE and immunoblotting for indicated proteins. The density of each protein band was divided by the average of the density of all bands from the same protein on the membrane. Fold changes were calculated by dividing each normalized ratio (protein to loading control) by the average of the ratios of the control lane (medium). Statistical graphics represents mean + SD (n = 3). ( C ) L929 cells retrovirally transfected with cDNA encoding mRFP-EGFP-rLC3 were transfected with non-targeting (siCtrl) or Ripk3 siRNAs (si Ripk3 ). 48 h post transfection, cells were left untreated (medium, M) or exposed to indicated treatments (10 ng/ml TNF [T], 30 µM QVD [Q], 20 nM bafilomycin A 1 [B]) for 3 h. Then cells were fixed and RFP and GFP fluorescence was analyzed by immunofluorescence microscopy. The colocalization intensity was analyzed using Pearson’s correlation coefficient using ImageJ software. Scale bar: 20 µm. ( D ) L929 cells were retrovirally transfected with cDNA encoding mCitrine-LC3B. Cells were left untreated (medium, M) or treated using 10 ng/ml TNF + 30 µM QVD (TQ) or EBSS with or without 20 nM bafilomycin A 1 (B) for indicated times. Cells were collected and mCitrine fluorescence intensity was measured by flow cytometry. The mean of fluorescence intensity for each sample was normalized to cells incubated in growth medium (M). Data represent mean + SD from two independent experiments. ( A-D ) Statistical analysis was done by repeated measures two-way ANOVA (corrected by Tukey’s multiple comparisons test) for A, B and D, and by ordinary one-way ANOVA (corrected by Tukey’s multiple comparisons test) for C. For C, statistical analysis was additionally performed using unpaired t test with Welch’s correction (TQ treatment of non-targeting vs. Ripk3 siRNA). For A and B, statistically significant differences are only indicated for 6 h; for D, statistically significant differences are only indicated for 6 h vs. 6 h + bafilomycin A 1 . Statistically significant differences to control (medium, M) are depicted as letters directly above the bars. * or a: P < 0.05, ** or b: P < 0.01, *** or c: P < 0.001, **** or d: P < 0.0001
    Figure Legend Snippet: Necroptosis inhibits lysosomal LC3 degradation. ( A ) L929 cells were left untreated (medium, M) or exposed to 30 µM QVD (Q), 20 nM bafilomycin A 1 (B), 10 ng/ml TNF (T), 10 ng/ml TNF + 30 µM QVD with or without 20 nM bafilomycin A 1 (TQ or TQB) for indicated times. Then, cells were lysed and cleared cellular lysates were subjected to SDS-PAGE and immunoblotting for indicated proteins. The density of each protein band was divided by the average of the density of all bands from the same protein on the membrane. Fold changes were calculated by dividing each normalized ratio (protein to loading control) by the average of the ratios of the control lane (medium). Statistical graphics represents mean + SD (n = 4). ( B ) L929 WT, ripk3 KO or MLKL KO cells were exposed to 10 ng/ml TNF and 30 µM QVD (TQ) for indicated times. Then, cells were lysed and cleared cellular lysates were subjected to SDS-PAGE and immunoblotting for indicated proteins. The density of each protein band was divided by the average of the density of all bands from the same protein on the membrane. Fold changes were calculated by dividing each normalized ratio (protein to loading control) by the average of the ratios of the control lane (medium). Statistical graphics represents mean + SD (n = 3). ( C ) L929 cells retrovirally transfected with cDNA encoding mRFP-EGFP-rLC3 were transfected with non-targeting (siCtrl) or Ripk3 siRNAs (si Ripk3 ). 48 h post transfection, cells were left untreated (medium, M) or exposed to indicated treatments (10 ng/ml TNF [T], 30 µM QVD [Q], 20 nM bafilomycin A 1 [B]) for 3 h. Then cells were fixed and RFP and GFP fluorescence was analyzed by immunofluorescence microscopy. The colocalization intensity was analyzed using Pearson’s correlation coefficient using ImageJ software. Scale bar: 20 µm. ( D ) L929 cells were retrovirally transfected with cDNA encoding mCitrine-LC3B. Cells were left untreated (medium, M) or treated using 10 ng/ml TNF + 30 µM QVD (TQ) or EBSS with or without 20 nM bafilomycin A 1 (B) for indicated times. Cells were collected and mCitrine fluorescence intensity was measured by flow cytometry. The mean of fluorescence intensity for each sample was normalized to cells incubated in growth medium (M). Data represent mean + SD from two independent experiments. ( A-D ) Statistical analysis was done by repeated measures two-way ANOVA (corrected by Tukey’s multiple comparisons test) for A, B and D, and by ordinary one-way ANOVA (corrected by Tukey’s multiple comparisons test) for C. For C, statistical analysis was additionally performed using unpaired t test with Welch’s correction (TQ treatment of non-targeting vs. Ripk3 siRNA). For A and B, statistically significant differences are only indicated for 6 h; for D, statistically significant differences are only indicated for 6 h vs. 6 h + bafilomycin A 1 . Statistically significant differences to control (medium, M) are depicted as letters directly above the bars. * or a: P < 0.05, ** or b: P < 0.01, *** or c: P < 0.001, **** or d: P < 0.0001

    Techniques Used: SDS Page, Western Blot, Transfection, Fluorescence, Immunofluorescence, Microscopy, Software, Flow Cytometry, Incubation

    Necroptosis induced by TNF destabilizes SNARE complexes and cleaves STX17 to block LC3 degradation. ( A ) L929 cells stably expressing GFP-SNAP29 were exposed to 10 ng/ml TNF and 30 µM QVD (TQ) for indicated times. Then, cells were lysed and cleared cellular lysates were subjected to immunopurification using anti-GFP beads. Purified proteins were subjected to SDS-PAGE and analyzed by immunoblotting for indicated proteins. ( B ) L929 cells stably expressing GFP-SNAP29 were left untreated (medium, M) or exposed to 10 ng/ml TNF + 30 µM QVD (TQ), TQ plus 5 µM GSK’872 (TQG), or TQ plus 5 µM necrostatin-1 (TQN) for 4 h. Then, cells were lysed and cleared cellular lysates were subjected to immunopurification using anti-GFP beads. Purified proteins were subjected to SDS-PAGE and analyzed by immunoblotting for indicated proteins. ( C ) L929 cells were left untreated (medium, M) or exposed to 30 µM QVD (Q), 20 nM bafilomycin A 1 (B), 10 ng/ml TNF (T), 10 ng/ml TNF + 30 µM QVD with or without 20 nM bafilomycin A1 (TQ or TQB) for indicated times. Then, cells were lysed and cleared cellular lysates were subjected to SDS-PAGE and immunoblotting for STX17 and ACTB. ( D ) L929 WT, ripk 3 KO or mlkl KO cells were left untreated or exposed to 10 ng/ml TNF + 30 µM QVD (TQ) for 4 h. Then, cells were lysed and cleared cellular lysates were subjected to SDS-PAGE and immunoblotting for STX17, RIPK3, MLKL, and GAPDH. ( E ) L929 cells were transfected with non-targeting (siCtrl) or Prkaa1/Prkaa2 siRNAs (si Prkaa1/ si Prkaa2 ). 48 h post transfection, cells were left untreated (medium, M) or exposed to 10 ng/ml TNF + 30 µM QVD (TQ) with or without 5 µM GSK’872 (G) for 4 h. Then, cells were lysed and cleared cellular lysates were subjected to SDS-PAGE and analyzed by immunoblotting for indicated proteins. The density of each protein band was divided by the average of the density of all bands from the same protein on the membrane. Fold changes were calculated by dividing each normalized ratio (protein to loading control) by the average of the ratios of the control lane (siCtrl, medium). Statistical graphics represents mean + SD (n = 3). Statistical analysis was done by ordinary two-way ANOVA (corrected by Tukey’s multiple comparisons test). Statistically significant differences are only indicated for TQ and TQG (scr vs. Prkaa1/Prkaa2 siRNA) and for TQ vs. TQG (within each treatment). Statistically significant differences to control (siCtrl, medium) are depicted as letters directly above the bars. ** or b: P < 0.01, *** or c: P < 0.001, ns: non-significant. ( F ) L929 cells were left untransfected or transiently transfected with cDNA encoding 3xFLAG-MmRIPK3 for 24 h. Then untransfected cells were left untreated or exposed to 10 ng/ml TNF + 30 µM QVD (TQ) for 3 h. Cells were lysed and cleared cellular lysates were subjected to SDS-PAGE and immunoblotting for indicated proteins
    Figure Legend Snippet: Necroptosis induced by TNF destabilizes SNARE complexes and cleaves STX17 to block LC3 degradation. ( A ) L929 cells stably expressing GFP-SNAP29 were exposed to 10 ng/ml TNF and 30 µM QVD (TQ) for indicated times. Then, cells were lysed and cleared cellular lysates were subjected to immunopurification using anti-GFP beads. Purified proteins were subjected to SDS-PAGE and analyzed by immunoblotting for indicated proteins. ( B ) L929 cells stably expressing GFP-SNAP29 were left untreated (medium, M) or exposed to 10 ng/ml TNF + 30 µM QVD (TQ), TQ plus 5 µM GSK’872 (TQG), or TQ plus 5 µM necrostatin-1 (TQN) for 4 h. Then, cells were lysed and cleared cellular lysates were subjected to immunopurification using anti-GFP beads. Purified proteins were subjected to SDS-PAGE and analyzed by immunoblotting for indicated proteins. ( C ) L929 cells were left untreated (medium, M) or exposed to 30 µM QVD (Q), 20 nM bafilomycin A 1 (B), 10 ng/ml TNF (T), 10 ng/ml TNF + 30 µM QVD with or without 20 nM bafilomycin A1 (TQ or TQB) for indicated times. Then, cells were lysed and cleared cellular lysates were subjected to SDS-PAGE and immunoblotting for STX17 and ACTB. ( D ) L929 WT, ripk 3 KO or mlkl KO cells were left untreated or exposed to 10 ng/ml TNF + 30 µM QVD (TQ) for 4 h. Then, cells were lysed and cleared cellular lysates were subjected to SDS-PAGE and immunoblotting for STX17, RIPK3, MLKL, and GAPDH. ( E ) L929 cells were transfected with non-targeting (siCtrl) or Prkaa1/Prkaa2 siRNAs (si Prkaa1/ si Prkaa2 ). 48 h post transfection, cells were left untreated (medium, M) or exposed to 10 ng/ml TNF + 30 µM QVD (TQ) with or without 5 µM GSK’872 (G) for 4 h. Then, cells were lysed and cleared cellular lysates were subjected to SDS-PAGE and analyzed by immunoblotting for indicated proteins. The density of each protein band was divided by the average of the density of all bands from the same protein on the membrane. Fold changes were calculated by dividing each normalized ratio (protein to loading control) by the average of the ratios of the control lane (siCtrl, medium). Statistical graphics represents mean + SD (n = 3). Statistical analysis was done by ordinary two-way ANOVA (corrected by Tukey’s multiple comparisons test). Statistically significant differences are only indicated for TQ and TQG (scr vs. Prkaa1/Prkaa2 siRNA) and for TQ vs. TQG (within each treatment). Statistically significant differences to control (siCtrl, medium) are depicted as letters directly above the bars. ** or b: P < 0.01, *** or c: P < 0.001, ns: non-significant. ( F ) L929 cells were left untransfected or transiently transfected with cDNA encoding 3xFLAG-MmRIPK3 for 24 h. Then untransfected cells were left untreated or exposed to 10 ng/ml TNF + 30 µM QVD (TQ) for 3 h. Cells were lysed and cleared cellular lysates were subjected to SDS-PAGE and immunoblotting for indicated proteins

    Techniques Used: Blocking Assay, Stable Transfection, Expressing, Immu-Puri, Purification, SDS Page, Western Blot, Transfection

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    ProSci Incorporated mouse ripk3
    (A) Cultured <t>MCF7/TO-RIPK3</t> and HeLa/TO-RIPK3 cells were treated with DMSO or Dox(1μg/ml) induction for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells. The cell lysates were analyzed by western blotting using antibodies against RIPK3 or β-actin (lower panel). (B) Cultured MCF7/TO-RIPK3, KGN/TO-RIPK3, and HeLa/TO-RIPK3 cells were treated with DMSO, Dox, or Dox plus TSZ for 36 hours. Cell viability was determined by measuring cellular ATP levels. The data are represented as the mean ± SD of triplicate wells. (C) Cultured MCF7/TO-RIPK3 cells were treated with DMSO or Dox, plus the indicated agents for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells. The cell lysates were analyzed by western blotting using antibodies against RIPK3 or β-actin (lower panel). 20 μM Z, pan-caspase inhibitor z-VAD; 2 μM RIPA-56, RIPK1 inhibitor; 2 μM NSA, MLKL inhibitor. (D) Cultured MCF7 cells were infected with lentiviruses encoding RIPK3(WT), RIPK3(AAAA), RIPK3(K50A), and RIPK3(K50A)+GSK’872 plus Z for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells. *** P <0.001. The lysates were measured by western blotting using antibodies against RIPK3 or β-actin as indicated (lower panel). GSK’872, RIPK3 inhibitor. The online version of this article includes the following figure supplement(s) for : Figure supplement 1 . RIPK3-induced apoptosis in human granulosa lutein cells (KGN).
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    1) Product Images from "A phosphorylation of RIPK3 kinase initiates an intracellular apoptotic pathway that promotes corpus luteum regression"

    Article Title: A phosphorylation of RIPK3 kinase initiates an intracellular apoptotic pathway that promotes corpus luteum regression

    Journal: bioRxiv

    doi: 10.1101/2021.02.14.431152

    (A) Cultured MCF7/TO-RIPK3 and HeLa/TO-RIPK3 cells were treated with DMSO or Dox(1μg/ml) induction for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells. The cell lysates were analyzed by western blotting using antibodies against RIPK3 or β-actin (lower panel). (B) Cultured MCF7/TO-RIPK3, KGN/TO-RIPK3, and HeLa/TO-RIPK3 cells were treated with DMSO, Dox, or Dox plus TSZ for 36 hours. Cell viability was determined by measuring cellular ATP levels. The data are represented as the mean ± SD of triplicate wells. (C) Cultured MCF7/TO-RIPK3 cells were treated with DMSO or Dox, plus the indicated agents for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells. The cell lysates were analyzed by western blotting using antibodies against RIPK3 or β-actin (lower panel). 20 μM Z, pan-caspase inhibitor z-VAD; 2 μM RIPA-56, RIPK1 inhibitor; 2 μM NSA, MLKL inhibitor. (D) Cultured MCF7 cells were infected with lentiviruses encoding RIPK3(WT), RIPK3(AAAA), RIPK3(K50A), and RIPK3(K50A)+GSK’872 plus Z for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells. *** P <0.001. The lysates were measured by western blotting using antibodies against RIPK3 or β-actin as indicated (lower panel). GSK’872, RIPK3 inhibitor. The online version of this article includes the following figure supplement(s) for : Figure supplement 1 . RIPK3-induced apoptosis in human granulosa lutein cells (KGN).
    Figure Legend Snippet: (A) Cultured MCF7/TO-RIPK3 and HeLa/TO-RIPK3 cells were treated with DMSO or Dox(1μg/ml) induction for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells. The cell lysates were analyzed by western blotting using antibodies against RIPK3 or β-actin (lower panel). (B) Cultured MCF7/TO-RIPK3, KGN/TO-RIPK3, and HeLa/TO-RIPK3 cells were treated with DMSO, Dox, or Dox plus TSZ for 36 hours. Cell viability was determined by measuring cellular ATP levels. The data are represented as the mean ± SD of triplicate wells. (C) Cultured MCF7/TO-RIPK3 cells were treated with DMSO or Dox, plus the indicated agents for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells. The cell lysates were analyzed by western blotting using antibodies against RIPK3 or β-actin (lower panel). 20 μM Z, pan-caspase inhibitor z-VAD; 2 μM RIPA-56, RIPK1 inhibitor; 2 μM NSA, MLKL inhibitor. (D) Cultured MCF7 cells were infected with lentiviruses encoding RIPK3(WT), RIPK3(AAAA), RIPK3(K50A), and RIPK3(K50A)+GSK’872 plus Z for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells. *** P <0.001. The lysates were measured by western blotting using antibodies against RIPK3 or β-actin as indicated (lower panel). GSK’872, RIPK3 inhibitor. The online version of this article includes the following figure supplement(s) for : Figure supplement 1 . RIPK3-induced apoptosis in human granulosa lutein cells (KGN).

    Techniques Used: Cell Culture, Western Blot, Infection

    Cultured KGN/TO-RIPK3 cells were treated with DMSO or Dox(1μg/ml) induction for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells. The cell lysates were analyzed by western blotting using antibodies against RIPK3 or β-actin (lower panel).
    Figure Legend Snippet: Cultured KGN/TO-RIPK3 cells were treated with DMSO or Dox(1μg/ml) induction for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells. The cell lysates were analyzed by western blotting using antibodies against RIPK3 or β-actin (lower panel).

    Techniques Used: Cell Culture, Western Blot

    (A) Cultured MCF7/TO-RIPK3 cells were treated with DMSO or Dox plus the indicated agent for 24 hours. The cells were then harvested, and RIPK3 was immunoprecipitated from the cell lysates using anti-Flag resin. The cell lysates and immunocomplexes were analyzed by western blotting using antibodies as indicated. (B - E ) Cultured MCF7/TO-RIPK3(wild type (WT), RIPK1 -/- , Caspase8 -/- , FADD -/- , and cFLIF -/- ) cells were treated with DMSO or Dox induction for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells. The cell lysates were analyzed by western blotting using antibodies against RIPK1, Caspase8, FADD, cFLIP or β-actin (lower panel).
    Figure Legend Snippet: (A) Cultured MCF7/TO-RIPK3 cells were treated with DMSO or Dox plus the indicated agent for 24 hours. The cells were then harvested, and RIPK3 was immunoprecipitated from the cell lysates using anti-Flag resin. The cell lysates and immunocomplexes were analyzed by western blotting using antibodies as indicated. (B - E ) Cultured MCF7/TO-RIPK3(wild type (WT), RIPK1 -/- , Caspase8 -/- , FADD -/- , and cFLIF -/- ) cells were treated with DMSO or Dox induction for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells. The cell lysates were analyzed by western blotting using antibodies against RIPK1, Caspase8, FADD, cFLIP or β-actin (lower panel).

    Techniques Used: Cell Culture, Immunoprecipitation, Western Blot

    (A) Cultured MCF7/TO-RIPK3 and HeLa/TO-RIPK3 cells were treated with Dox plus z-VAD for 24 hours. RIPK3 was immunoprecipitated from the cell lysates using anti-Flag resin. The RIPK3 bands were excised and subjected to mass spectrometry analysis. RIPK3 specific phosphorylation site in MCF7/TO-RIPK3 cells is highlighted in red. (B) Cultured KGN/TO-RIPK3, MCF7/TO-RIPK3, and HeLa/TO-RIPK3 cells were treated with Dox plus z-VAD for 24 hours. The lysates were analyzed by western blotting using antibodies against the phopho-Serine164/theronine165 of RIPK3, Flag (RIPK3), and β-actin as indicated. (C) Cultured MCF7 stably transfected with either wild type RIPK3 (WT) or kinase-dead mutant (D160N) cells under the control of Dox-inducible promoter were treated with DMSO(-) or Dox plus z-VAD for 24 hours. The lysates were analyzed by western blotting using antibodies against the phopho-Serine164/theronine165 RIPK3, Flag (RIPK3), and β-actin as indicated. (D) Cultured 293T cells were transfected with Vector (Vec), RIPK3(WT), RIPK3(D160N), RIPK3(AAAA) (RIPK3-AAAA, residues 459-462 mutated to AAAA) and RIPK3(S164D/T165E) for 24 hours. The level of phospho-S227-RIPK3 and RIPK3 were measured by western blotting. (E and F) Cultured MCF7 (E) and KGN (F) cells were infected with lentiviruses encoding RIPK3(WT), RIPK3(S164D/T165E), RIPK3(S164A/T165A) and RIPK3(AAAA) plus z-VAD for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells. *** P <0.001. The lysates were measured by western blotting using antibodies against RIPK3 or β-actin as indicated (lower panel). (G) Cultured HeLa cells were infected with lentiviruses encoding RIPK3(WT), RIPK3(D160N), RIPK3(AAAA), and RIPK3(S164D/T165E) plus z-VAD for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells. ** P <0.01, *** P <0.001. The expressed RIPK3 in the cell lysates were measured by western blotting using antibodies against RIPK3 or β-actin as indicated (lower panel). Vector (Vec, control viruses) (H) Cultured HeLa cells were transfected with Flag-tagged RIPK3(WT), RIPK3(D160N), and RIPK3(S164D/T165E) for 24 hours. RIPK3 was immunoprecipitated using anti-Flag resin. The lysates and immunocomplexes were analyzed by western blotting using antibodies against RIPK1, Caspase-8, FADD, and RIPK3 as indicated. (I) Cultured MCF7/TO-RIPK3 and MCF7/TO-RIPK3(S164A/T165A) cells were treated with Dox plus Z for 24 hours. The cells were then harvested, and RIPK3 was immunoprecipitated from the cell lysates using anti-Flag resin. The cell lysates and immunocomplexes were analyzed by western blotting using antibodies as indicated. The online version of this article includes the following figure supplement(s) for figure 3: Figure supplement 1 . Characterization of RIPK3 auto-phosphorylation sites. Figure supplement 2 . The phosphorylation site of RIPK3 is conserved among different mammalian species.
    Figure Legend Snippet: (A) Cultured MCF7/TO-RIPK3 and HeLa/TO-RIPK3 cells were treated with Dox plus z-VAD for 24 hours. RIPK3 was immunoprecipitated from the cell lysates using anti-Flag resin. The RIPK3 bands were excised and subjected to mass spectrometry analysis. RIPK3 specific phosphorylation site in MCF7/TO-RIPK3 cells is highlighted in red. (B) Cultured KGN/TO-RIPK3, MCF7/TO-RIPK3, and HeLa/TO-RIPK3 cells were treated with Dox plus z-VAD for 24 hours. The lysates were analyzed by western blotting using antibodies against the phopho-Serine164/theronine165 of RIPK3, Flag (RIPK3), and β-actin as indicated. (C) Cultured MCF7 stably transfected with either wild type RIPK3 (WT) or kinase-dead mutant (D160N) cells under the control of Dox-inducible promoter were treated with DMSO(-) or Dox plus z-VAD for 24 hours. The lysates were analyzed by western blotting using antibodies against the phopho-Serine164/theronine165 RIPK3, Flag (RIPK3), and β-actin as indicated. (D) Cultured 293T cells were transfected with Vector (Vec), RIPK3(WT), RIPK3(D160N), RIPK3(AAAA) (RIPK3-AAAA, residues 459-462 mutated to AAAA) and RIPK3(S164D/T165E) for 24 hours. The level of phospho-S227-RIPK3 and RIPK3 were measured by western blotting. (E and F) Cultured MCF7 (E) and KGN (F) cells were infected with lentiviruses encoding RIPK3(WT), RIPK3(S164D/T165E), RIPK3(S164A/T165A) and RIPK3(AAAA) plus z-VAD for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells. *** P <0.001. The lysates were measured by western blotting using antibodies against RIPK3 or β-actin as indicated (lower panel). (G) Cultured HeLa cells were infected with lentiviruses encoding RIPK3(WT), RIPK3(D160N), RIPK3(AAAA), and RIPK3(S164D/T165E) plus z-VAD for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells. ** P <0.01, *** P <0.001. The expressed RIPK3 in the cell lysates were measured by western blotting using antibodies against RIPK3 or β-actin as indicated (lower panel). Vector (Vec, control viruses) (H) Cultured HeLa cells were transfected with Flag-tagged RIPK3(WT), RIPK3(D160N), and RIPK3(S164D/T165E) for 24 hours. RIPK3 was immunoprecipitated using anti-Flag resin. The lysates and immunocomplexes were analyzed by western blotting using antibodies against RIPK1, Caspase-8, FADD, and RIPK3 as indicated. (I) Cultured MCF7/TO-RIPK3 and MCF7/TO-RIPK3(S164A/T165A) cells were treated with Dox plus Z for 24 hours. The cells were then harvested, and RIPK3 was immunoprecipitated from the cell lysates using anti-Flag resin. The cell lysates and immunocomplexes were analyzed by western blotting using antibodies as indicated. The online version of this article includes the following figure supplement(s) for figure 3: Figure supplement 1 . Characterization of RIPK3 auto-phosphorylation sites. Figure supplement 2 . The phosphorylation site of RIPK3 is conserved among different mammalian species.

    Techniques Used: Cell Culture, Immunoprecipitation, Mass Spectrometry, Western Blot, Stable Transfection, Transfection, Mutagenesis, Plasmid Preparation, Infection

    (A) Cultured MCF7/TO-RIPK3 and MCF7/TO-RIPK3(S164A/T165A) cells were treated with DMSO (-) or Dox plus z-VAD for 24 hours. The lysates were analyzed by western blotting using antibodies against the phopho-Serine164/theronine165 of RIPK3, Flag (RIPK3), and β-actin as indicated. (B) Cultured KGN7/TO-RIPK3 and KGN/TO-RIPK3(S164A/T165A) cells were treated with DMSO or Dox plus z-VAD for 24 hours. The lysates were analyzed by western blotting using antibodies against the phopho-Serine164/theronine165 of RIPK3, Flag (RIPK3), and β-actin as indicated. ( C) Cultured MCF7/TO-RIPK3 and MCF7/TO-RIPK3(K50A) cells were treated with DMSO (-) or Dox plus z-VAD for 24 hours. The lysates were analyzed by western blotting using antibodies against the phopho-Serine164/theronine165 of RIPK3, Flag (RIPK3), and β-actin as indicated. (D) Cultured HeLa cells were infected with lentiviruses encoding wild type RIPK3(WT), and mutant RIPK3 including RIPK3(D160N), RIPK3(S164D/T165E), RIPK3(S164E), RIPK3(T165E), RIPK3(S164A/T165A), RIPK3(S164A), RIPK3(T165A) and RIPK3(AAAA) and treated with TSZ for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data represented as the mean ± SD of triplicate wells. ** P <0.01. The levels of expressed RIPK3 in the cell lysates were measured by western blotting (lower panel). (E) RIPK3 single site (S164E) mutation blocks auto-phosphorylation. 293T cells were transfected with Flag-tagged RIPK3(WT), RIPK3(D160N), RIPK3(S164D/T165E), RIPK3(S164E), RIPK3(T165E), RIPK3(S164A/T165A), RIPK3(S164A), RIPK3(T165A) and RIPK3(AAAA) for 24 hours. The level of p-S227-RIPK3 and RIPK3 were measured by western blotting. (F and G) Cultured MCF7 (E) and KGN (F) cells were infected with lentiviruses encoding wild type RIPK3(WT), and mutant forms of RIPK3(S164A/T165A), RIPK3(S164A) and RIPK3(T165A) and treated with z-VAD as indicated for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells. *** P <0.001. The levels of RIPK3 in the cell lysates were measured by western blotting (lower panel). (H) Cultured HeLa cells were transfected with Flag-tagged wild type RIPK3(WT), and mutant forms of RIPK3(T165E), RIPK3(S164D/T165E), and RIPK3(S164E) for 24 hours. RIPK3 was immunoprecipitated from the cell lysates using anti-Flag resin. The lysates and immunocomplexes were analyzed by western blotting using antibodies against RIPK1, caspase-8, RIPK3, and β-actin as indicated.
    Figure Legend Snippet: (A) Cultured MCF7/TO-RIPK3 and MCF7/TO-RIPK3(S164A/T165A) cells were treated with DMSO (-) or Dox plus z-VAD for 24 hours. The lysates were analyzed by western blotting using antibodies against the phopho-Serine164/theronine165 of RIPK3, Flag (RIPK3), and β-actin as indicated. (B) Cultured KGN7/TO-RIPK3 and KGN/TO-RIPK3(S164A/T165A) cells were treated with DMSO or Dox plus z-VAD for 24 hours. The lysates were analyzed by western blotting using antibodies against the phopho-Serine164/theronine165 of RIPK3, Flag (RIPK3), and β-actin as indicated. ( C) Cultured MCF7/TO-RIPK3 and MCF7/TO-RIPK3(K50A) cells were treated with DMSO (-) or Dox plus z-VAD for 24 hours. The lysates were analyzed by western blotting using antibodies against the phopho-Serine164/theronine165 of RIPK3, Flag (RIPK3), and β-actin as indicated. (D) Cultured HeLa cells were infected with lentiviruses encoding wild type RIPK3(WT), and mutant RIPK3 including RIPK3(D160N), RIPK3(S164D/T165E), RIPK3(S164E), RIPK3(T165E), RIPK3(S164A/T165A), RIPK3(S164A), RIPK3(T165A) and RIPK3(AAAA) and treated with TSZ for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data represented as the mean ± SD of triplicate wells. ** P <0.01. The levels of expressed RIPK3 in the cell lysates were measured by western blotting (lower panel). (E) RIPK3 single site (S164E) mutation blocks auto-phosphorylation. 293T cells were transfected with Flag-tagged RIPK3(WT), RIPK3(D160N), RIPK3(S164D/T165E), RIPK3(S164E), RIPK3(T165E), RIPK3(S164A/T165A), RIPK3(S164A), RIPK3(T165A) and RIPK3(AAAA) for 24 hours. The level of p-S227-RIPK3 and RIPK3 were measured by western blotting. (F and G) Cultured MCF7 (E) and KGN (F) cells were infected with lentiviruses encoding wild type RIPK3(WT), and mutant forms of RIPK3(S164A/T165A), RIPK3(S164A) and RIPK3(T165A) and treated with z-VAD as indicated for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells. *** P <0.001. The levels of RIPK3 in the cell lysates were measured by western blotting (lower panel). (H) Cultured HeLa cells were transfected with Flag-tagged wild type RIPK3(WT), and mutant forms of RIPK3(T165E), RIPK3(S164D/T165E), and RIPK3(S164E) for 24 hours. RIPK3 was immunoprecipitated from the cell lysates using anti-Flag resin. The lysates and immunocomplexes were analyzed by western blotting using antibodies against RIPK1, caspase-8, RIPK3, and β-actin as indicated.

    Techniques Used: Cell Culture, Western Blot, Infection, Mutagenesis, Transfection, Immunoprecipitation

    (A) The cell lysates from cultured HT29, HeLa, MCF7, and KGN cells were analyzed by western blotting using antibodies as indicated. (B and C) Cultured HeLa-RIPK3, MCF7/TO-RIPK3, and KGN/TO-RIPK3 cells were treated with the indicated stimuli for 36 hours. Cell viability was determined by measuring cellular ATP levels. The data represented as the mean ± SD of triplicate wells. 17AAG, Hsp90 inhibitor. (D and E) HeLa/TO-RIPK3 cells were treated with the indicated stimuli for 36 hours. Cell viability was determined by measuring cellular ATP levels in (D). The data represented as the mean ± SD of triplicate wells. *** P <0.001. 24 hours after treatment, the cell lysates were analyzed by western blotting using antibodies against phopho-Serine164/Theronine165 of RIPK3, RIPK3, and β-actin as indicated in (E). (F and G) Cultured MCF7/TO-RIPK3 cells co-transfected with HSP90 and CDC37 as indicated were treated with DMSO or Dox for 36 hours. Cell viability was determined by measuring cellular ATP levels in (F). The data are represented as the mean ± SD of triplicate wells. *** P <0.001. 24 hours after treatment, the cell lysates were analyzed by western blotting using antibodies against phopho-Serine164/Theronine165 of RIPK3, RIPK3, Hsp90, CDC37, and β-actin as indicated in (G). (H) Cultured HeLa/TO-RIPK3 cells were treated with Dox or Dox plus 17AAG for 24 hours. The cells were then harvested, and RIPK3 was immunoprecipitated from the cell lysates using anti-Flag resin. The cell lysates and immunocomplexes were analyzed by western blotting using antibodies as indicated. (I) Cultured HeLa/TO-RIPK3 cells were treated with Dox or Dox plus 17AAG for 24 hours. Immunofluorescence of the cells with Flag-RIPK3 (red) antibody. Counterstaining with DAPI (blue). Scale bar, 10 μm. Higher-power views (right panels) were acquired from the selected boxed areas from the left panel. (J) Cultured MCF7/TO-RIPK3 cells co-transfected with HSP90 and CDC37 as indicated were treated with Dox for 24 hours. Immunofluorescence of the cells with Flag-RIPK3 (red) antibody. Counterstaining with DAPI (blue). Scale bar, 10 μm. Higher-power views (right panels) were acquired from the selected boxed areas from the left panel. The online version of this article includes the following figure supplement(s) for figure 4: Figure supplement 1 . Hsp90/CDC37 chaperone determines the necroptotic or apoptotic function of RIPK3 kinase. Figure supplement 2 . RIPK3 form amyloid-like structure in MCF7 and KGN cells. Figure supplement 3 . Hsp90/CDC37 chaperone protein levels was low in corpus luteum and corpus albicans.
    Figure Legend Snippet: (A) The cell lysates from cultured HT29, HeLa, MCF7, and KGN cells were analyzed by western blotting using antibodies as indicated. (B and C) Cultured HeLa-RIPK3, MCF7/TO-RIPK3, and KGN/TO-RIPK3 cells were treated with the indicated stimuli for 36 hours. Cell viability was determined by measuring cellular ATP levels. The data represented as the mean ± SD of triplicate wells. 17AAG, Hsp90 inhibitor. (D and E) HeLa/TO-RIPK3 cells were treated with the indicated stimuli for 36 hours. Cell viability was determined by measuring cellular ATP levels in (D). The data represented as the mean ± SD of triplicate wells. *** P <0.001. 24 hours after treatment, the cell lysates were analyzed by western blotting using antibodies against phopho-Serine164/Theronine165 of RIPK3, RIPK3, and β-actin as indicated in (E). (F and G) Cultured MCF7/TO-RIPK3 cells co-transfected with HSP90 and CDC37 as indicated were treated with DMSO or Dox for 36 hours. Cell viability was determined by measuring cellular ATP levels in (F). The data are represented as the mean ± SD of triplicate wells. *** P <0.001. 24 hours after treatment, the cell lysates were analyzed by western blotting using antibodies against phopho-Serine164/Theronine165 of RIPK3, RIPK3, Hsp90, CDC37, and β-actin as indicated in (G). (H) Cultured HeLa/TO-RIPK3 cells were treated with Dox or Dox plus 17AAG for 24 hours. The cells were then harvested, and RIPK3 was immunoprecipitated from the cell lysates using anti-Flag resin. The cell lysates and immunocomplexes were analyzed by western blotting using antibodies as indicated. (I) Cultured HeLa/TO-RIPK3 cells were treated with Dox or Dox plus 17AAG for 24 hours. Immunofluorescence of the cells with Flag-RIPK3 (red) antibody. Counterstaining with DAPI (blue). Scale bar, 10 μm. Higher-power views (right panels) were acquired from the selected boxed areas from the left panel. (J) Cultured MCF7/TO-RIPK3 cells co-transfected with HSP90 and CDC37 as indicated were treated with Dox for 24 hours. Immunofluorescence of the cells with Flag-RIPK3 (red) antibody. Counterstaining with DAPI (blue). Scale bar, 10 μm. Higher-power views (right panels) were acquired from the selected boxed areas from the left panel. The online version of this article includes the following figure supplement(s) for figure 4: Figure supplement 1 . Hsp90/CDC37 chaperone determines the necroptotic or apoptotic function of RIPK3 kinase. Figure supplement 2 . RIPK3 form amyloid-like structure in MCF7 and KGN cells. Figure supplement 3 . Hsp90/CDC37 chaperone protein levels was low in corpus luteum and corpus albicans.

    Techniques Used: Cell Culture, Western Blot, Transfection, Immunoprecipitation, Immunofluorescence

    (A) HeLa/TO-RIPK3 and HeLa/TO-RIPK3-shRNA-HSP90 cells were treated with the indicated stimuli for 36 hours. Cell viability was determined by measuring cellular ATP levels. The data represented as the mean ± SD of triplicate wells. *** P <0.001. The cell lysates were analyzed by western blotting using antibodies as indicated (right panel). (B) L929 cells were treated with the indicated stimuli for 5 hours. Cell viability was determined by measuring cellular ATP levels. The data represented as the mean ± SD of triplicate wells. (C) L929( Ripk3 -/- )/TO-RIPK3 and L929( Ripk3 -/- )/TO-RIPK3-shRNA-HSP90 cells were treated with the indicated stimuli for 36 hours. Cell viability was determined by measuring cellular ATP levels. The data represented as the mean ± SD of triplicate wells. *** P < 0.001. The cell lysates were analyzed by western blotting using antibodies as indicated (right panel). (D and E ) L929( Ripk3 -/- )/TO-RIPK3 cells were treated with the indicated stimuli for 36 hours. Cell viability was determined by measuring cellular ATP levels in (D). The data represented as the mean ± SD of triplicate wells. ** P <0.01, *** P <0.001. 24 hours after treatment, the cell lysates were analyzed by western blotting using antibodies against phopho-Serine165/Theronine166 of RIPK3, RIPK3, and β-actin as indicated in (E).
    Figure Legend Snippet: (A) HeLa/TO-RIPK3 and HeLa/TO-RIPK3-shRNA-HSP90 cells were treated with the indicated stimuli for 36 hours. Cell viability was determined by measuring cellular ATP levels. The data represented as the mean ± SD of triplicate wells. *** P <0.001. The cell lysates were analyzed by western blotting using antibodies as indicated (right panel). (B) L929 cells were treated with the indicated stimuli for 5 hours. Cell viability was determined by measuring cellular ATP levels. The data represented as the mean ± SD of triplicate wells. (C) L929( Ripk3 -/- )/TO-RIPK3 and L929( Ripk3 -/- )/TO-RIPK3-shRNA-HSP90 cells were treated with the indicated stimuli for 36 hours. Cell viability was determined by measuring cellular ATP levels. The data represented as the mean ± SD of triplicate wells. *** P < 0.001. The cell lysates were analyzed by western blotting using antibodies as indicated (right panel). (D and E ) L929( Ripk3 -/- )/TO-RIPK3 cells were treated with the indicated stimuli for 36 hours. Cell viability was determined by measuring cellular ATP levels in (D). The data represented as the mean ± SD of triplicate wells. ** P <0.01, *** P <0.001. 24 hours after treatment, the cell lysates were analyzed by western blotting using antibodies against phopho-Serine165/Theronine166 of RIPK3, RIPK3, and β-actin as indicated in (E).

    Techniques Used: shRNA, Western Blot

    (A) Alignment of amino acid sequences of RIPK3 orthologs in five mammalian species. Amino acid residues conserved in 80% or more of the sequences are shaded in black. The putative phosphorylation residues are denoted by asterisks (*). (B) Cultured 293T cells were transfected with Vector (Vec), mouse RIPK3(WT), RIPK3(S165D/T166E), and RIPK3(S165A/T165A) for 24 hours. The level of phospho-S232-RIPK3 and RIPK3 were measured by western blotting. (C) Cultured mouse sarcoma cells L929( Ripk3 -/- ) were transfected with Vector, wild type mouse RIPK3, and mutant forms of mRIPK3(D161N), and mRIPK3(S165D/T166E) and treated with z-VAD or TSZ as indicated for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data represented as the mean ± SD of triplicate wells. The lysates were analyzed by western blotting using antibodies as indicated (lower panel).
    Figure Legend Snippet: (A) Alignment of amino acid sequences of RIPK3 orthologs in five mammalian species. Amino acid residues conserved in 80% or more of the sequences are shaded in black. The putative phosphorylation residues are denoted by asterisks (*). (B) Cultured 293T cells were transfected with Vector (Vec), mouse RIPK3(WT), RIPK3(S165D/T166E), and RIPK3(S165A/T165A) for 24 hours. The level of phospho-S232-RIPK3 and RIPK3 were measured by western blotting. (C) Cultured mouse sarcoma cells L929( Ripk3 -/- ) were transfected with Vector, wild type mouse RIPK3, and mutant forms of mRIPK3(D161N), and mRIPK3(S165D/T166E) and treated with z-VAD or TSZ as indicated for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data represented as the mean ± SD of triplicate wells. The lysates were analyzed by western blotting using antibodies as indicated (lower panel).

    Techniques Used: Cell Culture, Transfection, Plasmid Preparation, Western Blot, Mutagenesis

    (A) Cultured HeLa/TO-RIPK3 and MCF7/TO-RIPK3 cells were treated with DMSO or Dox plus TSZ for 24 hours. Immunofluorescence of the cells with Flag-RIPK3 (green) antibody. Counterstaining with DAPI (blue). Scale bar, 10 μm. Higher-power views (right panels) were acquired from the selected boxed areas from the left panel. (B) Cultured HT29 and KGN/TO-RIPK3 cells were treated with DMSO, TSZ, or Dox for 24 hours. Immunofluorescence of the cells with Flag-RIPK3 (green) antibody. Counterstaining with DAPI (blue). Scale bar, 10 μm. Higher-power views (right panels) were acquired from the selected boxed areas from the left panel. (C) Cultured HeLa/TO-RIPK3 and MCF7/TO-RIPK3 cells were treated with Dox plus Z for 24 hours. Immunofluorescence of the cells with Flag-RIPK3 (red) and p-S164/T165-RIPK3(green) antibody. Counterstaining with DAPI (blue). Scale bar, 10 μm. Higher-power views (right panels) were acquired from the selected boxed areas from the left panel. (D) Cultured MCF7/TO-RIPK3(WT, RIPK1 -/- , caspase-8 -/- and FADD -/- ) cells were treated with Dox plus Z for 24 hours. Immunofluorescence of the cells with Flag-RIPK3 (red) and p-S164/T165-RIPK3(green) antibody. Counterstaining with DAPI (blue). Scale bar, 10 μm. Higher-power views (right panels) were acquired from the selected boxed areas from the left panel. (E) Cultured MCF7/TO-RIPK3(WT, RIPK1 -/- , caspase-8 -/- and FADD -/- ) cells were treated with DMSO or Dox plus Z for 24 hours. The lysates were analyzed by western blotting using antibodies as indicated.
    Figure Legend Snippet: (A) Cultured HeLa/TO-RIPK3 and MCF7/TO-RIPK3 cells were treated with DMSO or Dox plus TSZ for 24 hours. Immunofluorescence of the cells with Flag-RIPK3 (green) antibody. Counterstaining with DAPI (blue). Scale bar, 10 μm. Higher-power views (right panels) were acquired from the selected boxed areas from the left panel. (B) Cultured HT29 and KGN/TO-RIPK3 cells were treated with DMSO, TSZ, or Dox for 24 hours. Immunofluorescence of the cells with Flag-RIPK3 (green) antibody. Counterstaining with DAPI (blue). Scale bar, 10 μm. Higher-power views (right panels) were acquired from the selected boxed areas from the left panel. (C) Cultured HeLa/TO-RIPK3 and MCF7/TO-RIPK3 cells were treated with Dox plus Z for 24 hours. Immunofluorescence of the cells with Flag-RIPK3 (red) and p-S164/T165-RIPK3(green) antibody. Counterstaining with DAPI (blue). Scale bar, 10 μm. Higher-power views (right panels) were acquired from the selected boxed areas from the left panel. (D) Cultured MCF7/TO-RIPK3(WT, RIPK1 -/- , caspase-8 -/- and FADD -/- ) cells were treated with Dox plus Z for 24 hours. Immunofluorescence of the cells with Flag-RIPK3 (red) and p-S164/T165-RIPK3(green) antibody. Counterstaining with DAPI (blue). Scale bar, 10 μm. Higher-power views (right panels) were acquired from the selected boxed areas from the left panel. (E) Cultured MCF7/TO-RIPK3(WT, RIPK1 -/- , caspase-8 -/- and FADD -/- ) cells were treated with DMSO or Dox plus Z for 24 hours. The lysates were analyzed by western blotting using antibodies as indicated.

    Techniques Used: Cell Culture, Immunofluorescence, Western Blot

    (A) Two guide RNA and donate oligo sequences of Ripk3(S165D/T166E) and Ripk3(S165A/T166A) knock-in mice. (B) Schematic of CRISPER-Cas9 strategy for the generation for Ripk3(S165D/T166E) and Ripk3(S165A/T166A) knock-in mice. The gene structure of RIPK3 and two guide RNA sequences targeting the exon 4 of RIPK3 were shown with the PAM sequences highlighted in red and blue. (C and D ) Macroscopic features (C) and body weights (D) of Ripk3 +/+ and Ripk3 ST-AA/ST-AA littermate mice at 14 days of age (n=10). The result from each individual animal was presented as an indicated dot. NS, not significant. (E and F ) Immunoblot of RIPK3 from lung extracts of 14 days old Ripk3 +/+ , Ripk3 ST-DE/ST-DE , and Ripk3 ST-AA/ST-AA littermates using antibodies against RIPK3 and GAPDH as indicated (n=3). (G) Histological analysis of brain, cerebellum, heart, kidney, and liver of Ripk3 +/+ and Ripk3 ST-DE/ST-DE littermate mice (n=5) at 14 days of age. Scale bar, 20 μm. PTC, Proximal tubular cell. (H) Cell viability measurement of bone marrow-derived macrophages from the Ripk3 +/+ and Ripk3 ST-AA/ST-AA littermate mice (n=3, 14 days) after treatment with the indicated necroptosis stimuli for 24 hours. Cell viability was determined by measuring cellular ATP levels. The data represented as the mean ± SD of triplicate wells.
    Figure Legend Snippet: (A) Two guide RNA and donate oligo sequences of Ripk3(S165D/T166E) and Ripk3(S165A/T166A) knock-in mice. (B) Schematic of CRISPER-Cas9 strategy for the generation for Ripk3(S165D/T166E) and Ripk3(S165A/T166A) knock-in mice. The gene structure of RIPK3 and two guide RNA sequences targeting the exon 4 of RIPK3 were shown with the PAM sequences highlighted in red and blue. (C and D ) Macroscopic features (C) and body weights (D) of Ripk3 +/+ and Ripk3 ST-AA/ST-AA littermate mice at 14 days of age (n=10). The result from each individual animal was presented as an indicated dot. NS, not significant. (E and F ) Immunoblot of RIPK3 from lung extracts of 14 days old Ripk3 +/+ , Ripk3 ST-DE/ST-DE , and Ripk3 ST-AA/ST-AA littermates using antibodies against RIPK3 and GAPDH as indicated (n=3). (G) Histological analysis of brain, cerebellum, heart, kidney, and liver of Ripk3 +/+ and Ripk3 ST-DE/ST-DE littermate mice (n=5) at 14 days of age. Scale bar, 20 μm. PTC, Proximal tubular cell. (H) Cell viability measurement of bone marrow-derived macrophages from the Ripk3 +/+ and Ripk3 ST-AA/ST-AA littermate mice (n=3, 14 days) after treatment with the indicated necroptosis stimuli for 24 hours. Cell viability was determined by measuring cellular ATP levels. The data represented as the mean ± SD of triplicate wells.

    Techniques Used: Knock-In, Western Blot, Derivative Assay

    (A and B ) Macroscopic features (A) and body weights (B) of Ripk3 +/+ and Ripk3 ST-DE/ST-DE littermate mice at 14 days of age (n≥9). The result from each individual animal is presented as an indicated dot. *** P <0.001. (C) Kaplan-Meier plot of survival of Ripk3 +/+ and Ripk3 ST-DE/ST-DE littermate mice (n=10 for each genotype) after birth within two months. *** P <0.001. (D) Histological analysis of large intestine, small intestine, lung, and spleen of Ripk3 +/+ and Ripk3 ST-DE/ST-DE littermate mice (n=5) at 14 days of age. Scale bar, 20 μm. (E and F) Representative immunohistochemistry (IHC) images of the large intestine, small intestine, lung, and spleen of Ripk3 +/+ and Ripk3 ST-DE/ST-DE littermate mice (n=5, 14 days) stained with a Cleaved-Caspase3 (C-C3) antibody in (E). C-C3 positive cells were counted in two fields per organ and quantified in (F). Scale bar, 10 μm. Data represent the mean ± s.e.m. ** P <0.01, *** P <0.001. (G) Cell viability measurement of bone marrow-derived macrophages from the Ripk3 +/+ and Ripk3 ST-DE/ST-DE littermate mice (n=3, 14 days) after treatment with the indicated Z-VAD or necroptosis stimuli for 24 hours. Cell viability was determined by measuring cellular ATP levels. The data are represented as the mean ± SD of triplicate wells. The online version of this article includes the following figure supplement(s) for figure 5: Figure supplement 1 . Generation of Ripk3 ST-DE/ST-DE and Ripk3 ST-AA/ST-AA mice.
    Figure Legend Snippet: (A and B ) Macroscopic features (A) and body weights (B) of Ripk3 +/+ and Ripk3 ST-DE/ST-DE littermate mice at 14 days of age (n≥9). The result from each individual animal is presented as an indicated dot. *** P <0.001. (C) Kaplan-Meier plot of survival of Ripk3 +/+ and Ripk3 ST-DE/ST-DE littermate mice (n=10 for each genotype) after birth within two months. *** P <0.001. (D) Histological analysis of large intestine, small intestine, lung, and spleen of Ripk3 +/+ and Ripk3 ST-DE/ST-DE littermate mice (n=5) at 14 days of age. Scale bar, 20 μm. (E and F) Representative immunohistochemistry (IHC) images of the large intestine, small intestine, lung, and spleen of Ripk3 +/+ and Ripk3 ST-DE/ST-DE littermate mice (n=5, 14 days) stained with a Cleaved-Caspase3 (C-C3) antibody in (E). C-C3 positive cells were counted in two fields per organ and quantified in (F). Scale bar, 10 μm. Data represent the mean ± s.e.m. ** P <0.01, *** P <0.001. (G) Cell viability measurement of bone marrow-derived macrophages from the Ripk3 +/+ and Ripk3 ST-DE/ST-DE littermate mice (n=3, 14 days) after treatment with the indicated Z-VAD or necroptosis stimuli for 24 hours. Cell viability was determined by measuring cellular ATP levels. The data are represented as the mean ± SD of triplicate wells. The online version of this article includes the following figure supplement(s) for figure 5: Figure supplement 1 . Generation of Ripk3 ST-DE/ST-DE and Ripk3 ST-AA/ST-AA mice.

    Techniques Used: Immunohistochemistry, Staining, Derivative Assay

    (A) Western blot analysis of RIPK1, RIPK3, and MLKL levels in perfused mouse ovary extracts of different ages. Each group is representative of at least 3 mice. (B) H&E and immunofluorescence (IF) imaging of an 8-month-old ovary. Two adjacent sections were analyzed. One section was stained with H&E, and the other was IF stained with a RIPK3 antibody (red) and DAPI (blue). Scale bar, 500 μm. Higher-power views of selected areas were acquired in a (primordia follicle), b (secondary follicle), c (corpus luteum), and d (corpus albicans) as indicated. PF, primary follicle; CL, corpus luteum; CA, corpus albicans. (C) Ovarian PGF 2α levels of wild-type mice (n=8) at the indicated age assayed by ELISA. Data represent the mean ± s.e.m. ** P <0.01, *** P <0.001. (D) Immunofluorescence images of a RIPK3 C-terminus HA-3×Flag knock-in mouse ovary (n=5; 12 months) stained with antibodies against prostaglandin F receptor (PTGFR, green) and Flag (red). Counterstaining with DAPI (blue). Scale bar, 500 μm. Higher-power views (right panels) were acquired from the indicated boxed area in the second lower left panel. CL, corpus luteum. CA, corpus albicans. Scale bar, 100 μm. (E) Western blot analysis of p-S164/T165-RIPK3 and RIPK3 levels in extracts from perfused ovaries prepared from mice at the indicated age. Each group is representative of at least 3 mice. (F) Immunofluorescence images of ovaries from Ripk3 +/+ and Ripk3 -/- mice (4 Month, 8 Month and 12 Month; n=3) at the indicated ages stained with the p-S164/T165-RIPK3 antibody (red). Counterstaining with DAPI (blue). Scale bar, 200 μm. Higher-power views (lower two panels) were acquired from the selected boxed areas from the upper panel. (CL), b (CL), c (CL, CA) and d (CL). F, follicle; CL, corpus luteum; CA, corpus albicans. Scale bar, 100 μm. The online version of this article includes the following figure supplement(s) for figure 6: Figure supplement 1 . Generation of RIPK3 C-terminus HA-3×Flag knock-in and Ptgfr -/- mice.
    Figure Legend Snippet: (A) Western blot analysis of RIPK1, RIPK3, and MLKL levels in perfused mouse ovary extracts of different ages. Each group is representative of at least 3 mice. (B) H&E and immunofluorescence (IF) imaging of an 8-month-old ovary. Two adjacent sections were analyzed. One section was stained with H&E, and the other was IF stained with a RIPK3 antibody (red) and DAPI (blue). Scale bar, 500 μm. Higher-power views of selected areas were acquired in a (primordia follicle), b (secondary follicle), c (corpus luteum), and d (corpus albicans) as indicated. PF, primary follicle; CL, corpus luteum; CA, corpus albicans. (C) Ovarian PGF 2α levels of wild-type mice (n=8) at the indicated age assayed by ELISA. Data represent the mean ± s.e.m. ** P <0.01, *** P <0.001. (D) Immunofluorescence images of a RIPK3 C-terminus HA-3×Flag knock-in mouse ovary (n=5; 12 months) stained with antibodies against prostaglandin F receptor (PTGFR, green) and Flag (red). Counterstaining with DAPI (blue). Scale bar, 500 μm. Higher-power views (right panels) were acquired from the indicated boxed area in the second lower left panel. CL, corpus luteum. CA, corpus albicans. Scale bar, 100 μm. (E) Western blot analysis of p-S164/T165-RIPK3 and RIPK3 levels in extracts from perfused ovaries prepared from mice at the indicated age. Each group is representative of at least 3 mice. (F) Immunofluorescence images of ovaries from Ripk3 +/+ and Ripk3 -/- mice (4 Month, 8 Month and 12 Month; n=3) at the indicated ages stained with the p-S164/T165-RIPK3 antibody (red). Counterstaining with DAPI (blue). Scale bar, 200 μm. Higher-power views (lower two panels) were acquired from the selected boxed areas from the upper panel. (CL), b (CL), c (CL, CA) and d (CL). F, follicle; CL, corpus luteum; CA, corpus albicans. Scale bar, 100 μm. The online version of this article includes the following figure supplement(s) for figure 6: Figure supplement 1 . Generation of RIPK3 C-terminus HA-3×Flag knock-in and Ptgfr -/- mice.

    Techniques Used: Western Blot, Immunofluorescence, Imaging, Staining, Enzyme-linked Immunosorbent Assay, Knock-In

    (A) Schematic of CRISPER-Cas9 strategy for RIPK3 C-terminus HA-3×Flag knock-in mice. The gene structure of RIPK3 and guide RNA sequences targeting the Ripk3 were shown with the PAM sequences highlighted in red. (B) Western blotting analysis using protein extracts from the ovary of wild type, heterozygous knock-in, and homozygous knock-in mice generated as illustrated in (A). (C) Schematic of CRISPER-Cas9 strategy for the generation for Ptgfr -/- mice. The gene structure of PTGFR and two guide RNA sequences targeting the Ptgfr were shown with the PAM sequences highlighted in red. (D) Immunoblot of PTGFR from ovary extracts of 2-month old Ptgfr +/+ and Ptgfr -/- littermates using antibodies against PTGFR and GAPDH as indicated (n=3).
    Figure Legend Snippet: (A) Schematic of CRISPER-Cas9 strategy for RIPK3 C-terminus HA-3×Flag knock-in mice. The gene structure of RIPK3 and guide RNA sequences targeting the Ripk3 were shown with the PAM sequences highlighted in red. (B) Western blotting analysis using protein extracts from the ovary of wild type, heterozygous knock-in, and homozygous knock-in mice generated as illustrated in (A). (C) Schematic of CRISPER-Cas9 strategy for the generation for Ptgfr -/- mice. The gene structure of PTGFR and two guide RNA sequences targeting the Ptgfr were shown with the PAM sequences highlighted in red. (D) Immunoblot of PTGFR from ovary extracts of 2-month old Ptgfr +/+ and Ptgfr -/- littermates using antibodies against PTGFR and GAPDH as indicated (n=3).

    Techniques Used: Knock-In, Western Blot, Generated

    (A and B ) Immunofluorescence of ovary from wild type mice (8 Month; n=3) with RIPK3 (red) and HSP90 (green) antibody in (A). Higher-power views of selected areas were acquired in right panel. The HSP90/RIPK3 levels were quantified in (B). F, follicle. CA, corpus albicans. Scale bar, 100/200 μm. ( C and D ) Immunofluorescence of ovary from wild type mice (8 Month; n=3) with RIPK3 (red) and CDC37 (green) antibody in (C). Higher-power views of selected areas were acquired in right panel. The CDC37/RIPK3 levels were quantified in (D). F, follicle. CA, corpus albicans. Scale bar, 200 μm.
    Figure Legend Snippet: (A and B ) Immunofluorescence of ovary from wild type mice (8 Month; n=3) with RIPK3 (red) and HSP90 (green) antibody in (A). Higher-power views of selected areas were acquired in right panel. The HSP90/RIPK3 levels were quantified in (B). F, follicle. CA, corpus albicans. Scale bar, 100/200 μm. ( C and D ) Immunofluorescence of ovary from wild type mice (8 Month; n=3) with RIPK3 (red) and CDC37 (green) antibody in (C). Higher-power views of selected areas were acquired in right panel. The CDC37/RIPK3 levels were quantified in (D). F, follicle. CA, corpus albicans. Scale bar, 200 μm.

    Techniques Used: Immunofluorescence

    (A to C) Primary granulosal lutein cells (WT, Ripk3 -/- ) were isolated from 3-month-old mice ovaries. The cells were treated with Dinoprost Tromethamine (DT) at the indicated concentration for 36 hours in (A); with 1.5 μM DT at the indicated time in (B); or with 1.5 μM DT for 36 hours in (C). The cell lysates from the DT-treated cells were analyzed by western blotting using antibodies as indicated. (D and E) Ptgfr +/+ and Ptgfr -/- littermate female mice (n=16; 25-26 days) were given 7.5 IU pregnant mare serum gonadotropin (PMSG) intraperitoneally(IP) followed by 7.5 IU serum gonadotropin and chorionic gonadotropin (SCG) 46 hours later to synchronize ovulation. The animals were then injected with Dinoprost Tromethamine (DT) (10 micrograms, i.p.) or saline 24 hours post-ovulation. Ovaries were then collected 12 hours later and stained with anti-RIPK3 antibody (red) in (D). The ovary lysates were analyzed by western blotting using antibodies as indicated in (E). (*) indicates Corpus luteum. Counterstaining with DAPI (blue). Scale bar, 500 μm. (F and G) wild type(WT), Ripk3 -/- , Ripk3 S165A-T166A/S165A-T166A , Fadd -/- Mlkl -/- and Ptgfr -/- female mice (each group, n=16; 25-26 days) were treated as in (D and F). Ovaries from each group were then collected 24 hours after injecting with DT and stained with anti-cleaved-caspase3 antibody in (F). The Cleaved-Caspase3 + cells were counted in five fields per ovary Corpus luteum(CL) and quantified in (G). Scale bar, 20 μm. Data represent the mean ± s.e.m. ** P <0.01, *** P <0.001. (H) wild type female mice (n=3; 25-26 days) were treated as in (D and F). Ovaries were then collected 12 hours after injecting with DT and stained with anti-cleaved-caspase3(red) and p-S164/T165-RIPK3(green) antibody. Counterstaining with DAPI (blue). Scale bar, 100 μm. The online version of this article includes the following figure supplement(s) for figure 7: Figure supplement 1 . Prostaglandin F2alpha (PGF 2α ) stimulates RIPK3 expression through the MAPK pathway.
    Figure Legend Snippet: (A to C) Primary granulosal lutein cells (WT, Ripk3 -/- ) were isolated from 3-month-old mice ovaries. The cells were treated with Dinoprost Tromethamine (DT) at the indicated concentration for 36 hours in (A); with 1.5 μM DT at the indicated time in (B); or with 1.5 μM DT for 36 hours in (C). The cell lysates from the DT-treated cells were analyzed by western blotting using antibodies as indicated. (D and E) Ptgfr +/+ and Ptgfr -/- littermate female mice (n=16; 25-26 days) were given 7.5 IU pregnant mare serum gonadotropin (PMSG) intraperitoneally(IP) followed by 7.5 IU serum gonadotropin and chorionic gonadotropin (SCG) 46 hours later to synchronize ovulation. The animals were then injected with Dinoprost Tromethamine (DT) (10 micrograms, i.p.) or saline 24 hours post-ovulation. Ovaries were then collected 12 hours later and stained with anti-RIPK3 antibody (red) in (D). The ovary lysates were analyzed by western blotting using antibodies as indicated in (E). (*) indicates Corpus luteum. Counterstaining with DAPI (blue). Scale bar, 500 μm. (F and G) wild type(WT), Ripk3 -/- , Ripk3 S165A-T166A/S165A-T166A , Fadd -/- Mlkl -/- and Ptgfr -/- female mice (each group, n=16; 25-26 days) were treated as in (D and F). Ovaries from each group were then collected 24 hours after injecting with DT and stained with anti-cleaved-caspase3 antibody in (F). The Cleaved-Caspase3 + cells were counted in five fields per ovary Corpus luteum(CL) and quantified in (G). Scale bar, 20 μm. Data represent the mean ± s.e.m. ** P <0.01, *** P <0.001. (H) wild type female mice (n=3; 25-26 days) were treated as in (D and F). Ovaries were then collected 12 hours after injecting with DT and stained with anti-cleaved-caspase3(red) and p-S164/T165-RIPK3(green) antibody. Counterstaining with DAPI (blue). Scale bar, 100 μm. The online version of this article includes the following figure supplement(s) for figure 7: Figure supplement 1 . Prostaglandin F2alpha (PGF 2α ) stimulates RIPK3 expression through the MAPK pathway.

    Techniques Used: Isolation, Concentration Assay, Western Blot, Injection, Staining, Expressing

    (A) Primary granulosal lutein cells were isolated from the 3-month-old mice ovary. The cells were then treated with 1 μM DT or plus MAPK inhibitors PD-98059 (5 μM) and U0126 (5 μM) as indicated for 36 hours. The lysates were analyzed by western blotting using antibodies as indicated. (B) Primary granulosal lutein cells were isolated from 3-month-old mice ovaries. The cells were treated with 1 μM DT at the indicated time. The cell lysates from the DT-treated cells were analyzed by western blotting using antibodies as indicated. (C) Diagram of induction of corpus luteum regression in vivo . (D) Ripk3 +/+ and Ripk3 S165A-T166A/S165A-T166A and littermate female mice (n=3; 25-26 days) were given 7.5 IU pregnant mare serum gonadotropin (PMSG) intraperitoneally(IP) followed by 7.5 IU serum gonadotropin and chorionic gonadotropin (SCG) 46 hours later to synchronize ovulation. The animals were then injected with Dinoprost Tromethamine (DT) (10 micrograms, i.p.) or saline 24 hours post-ovulation. The ovary lysates were analyzed by western blotting using antibodies as indicated.
    Figure Legend Snippet: (A) Primary granulosal lutein cells were isolated from the 3-month-old mice ovary. The cells were then treated with 1 μM DT or plus MAPK inhibitors PD-98059 (5 μM) and U0126 (5 μM) as indicated for 36 hours. The lysates were analyzed by western blotting using antibodies as indicated. (B) Primary granulosal lutein cells were isolated from 3-month-old mice ovaries. The cells were treated with 1 μM DT at the indicated time. The cell lysates from the DT-treated cells were analyzed by western blotting using antibodies as indicated. (C) Diagram of induction of corpus luteum regression in vivo . (D) Ripk3 +/+ and Ripk3 S165A-T166A/S165A-T166A and littermate female mice (n=3; 25-26 days) were given 7.5 IU pregnant mare serum gonadotropin (PMSG) intraperitoneally(IP) followed by 7.5 IU serum gonadotropin and chorionic gonadotropin (SCG) 46 hours later to synchronize ovulation. The animals were then injected with Dinoprost Tromethamine (DT) (10 micrograms, i.p.) or saline 24 hours post-ovulation. The ovary lysates were analyzed by western blotting using antibodies as indicated.

    Techniques Used: Isolation, Western Blot, In Vivo, Injection

    mouse ripk3  (ProSci Incorporated)


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    ProSci Incorporated mouse 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.
    Mouse 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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    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

    mouse ripk3  (ProSci Incorporated)


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    ProSci Incorporated mouse 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.
    Mouse 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
    https://www.bioz.com/result/mouse ripk3/product/ProSci Incorporated
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse ripk3 - by Bioz Stars, 2023-06
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    Images

    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

    length mouse ripk3 isoform  (ProSci Incorporated)


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    ProSci Incorporated length mouse ripk3 isoform
    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.
    Length Mouse Ripk3 Isoform, 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 "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

    mouse ripk3  (ProSci Incorporated)


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    ProSci Incorporated mouse 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.
    Mouse 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 "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

    mouse ripk3  (ProSci Incorporated)


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    ProSci Incorporated mouse ripk3
    Mouse 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
    https://www.bioz.com/result/mouse ripk3/product/ProSci Incorporated
    Average 94 stars, based on 1 article reviews
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    mouse ripk3 - by Bioz Stars, 2023-06
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    anti mouse ripk3  (ProSci Incorporated)


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    ProSci Incorporated anti mouse ripk3
    a DAPK1 deficiency does not affect expressions of FADD, RIPK1, <t>RIPK3,</t> or MLKL in BMDMs. b , c Dapk1 − /− BMDMs exhibit increased cell death induction relative to WT upon zVAD+AT-406 treatment. WT and Dapk1 −/− BMDMs were stimulated with DMSO, AT-406 (0.6 μM, A), zVAD (20 μM, Z), Nec-1 (40 μM, N), or BV6 (0.5 μM, B), as indicated, for 18–20 h, before determining cell death according to release of ATP. d , e zVAD+TNF or zVAD+IFN-β treatments trigger increased necroptosis in Dapk1 −/− BMDMs. WT and Dapk1 −/− BMDMs were treated with zVAD + TNF (5 ng/ml) ( d ) or zVAD + IFN-β (5 ng/ml) ( e ) and then cell viability was determined. f High dose of AT-406 induces necroptosis. WT and Dapk1 −/− BMDMs were treated with AT-406 at the indicated dose, without or with Nec-1, and cell viability quantitated. Values are mean ± SD of triplicates in a single experiment. * P < 0.05, ** P < 0.01, *** P < 0.001 for unpaired t -test. Data have been repeated in two ( e , f) or three ( a – d ) independent experiments.
    Anti Mouse 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 "Tumor suppressor death-associated protein kinase 1 inhibits necroptosis by p38 MAPK activation"

    Article Title: Tumor suppressor death-associated protein kinase 1 inhibits necroptosis by p38 MAPK activation

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-020-2534-9

    a DAPK1 deficiency does not affect expressions of FADD, RIPK1, RIPK3, or MLKL in BMDMs. b , c Dapk1 − /− BMDMs exhibit increased cell death induction relative to WT upon zVAD+AT-406 treatment. WT and Dapk1 −/− BMDMs were stimulated with DMSO, AT-406 (0.6 μM, A), zVAD (20 μM, Z), Nec-1 (40 μM, N), or BV6 (0.5 μM, B), as indicated, for 18–20 h, before determining cell death according to release of ATP. d , e zVAD+TNF or zVAD+IFN-β treatments trigger increased necroptosis in Dapk1 −/− BMDMs. WT and Dapk1 −/− BMDMs were treated with zVAD + TNF (5 ng/ml) ( d ) or zVAD + IFN-β (5 ng/ml) ( e ) and then cell viability was determined. f High dose of AT-406 induces necroptosis. WT and Dapk1 −/− BMDMs were treated with AT-406 at the indicated dose, without or with Nec-1, and cell viability quantitated. Values are mean ± SD of triplicates in a single experiment. * P < 0.05, ** P < 0.01, *** P < 0.001 for unpaired t -test. Data have been repeated in two ( e , f) or three ( a – d ) independent experiments.
    Figure Legend Snippet: a DAPK1 deficiency does not affect expressions of FADD, RIPK1, RIPK3, or MLKL in BMDMs. b , c Dapk1 − /− BMDMs exhibit increased cell death induction relative to WT upon zVAD+AT-406 treatment. WT and Dapk1 −/− BMDMs were stimulated with DMSO, AT-406 (0.6 μM, A), zVAD (20 μM, Z), Nec-1 (40 μM, N), or BV6 (0.5 μM, B), as indicated, for 18–20 h, before determining cell death according to release of ATP. d , e zVAD+TNF or zVAD+IFN-β treatments trigger increased necroptosis in Dapk1 −/− BMDMs. WT and Dapk1 −/− BMDMs were treated with zVAD + TNF (5 ng/ml) ( d ) or zVAD + IFN-β (5 ng/ml) ( e ) and then cell viability was determined. f High dose of AT-406 induces necroptosis. WT and Dapk1 −/− BMDMs were treated with AT-406 at the indicated dose, without or with Nec-1, and cell viability quantitated. Values are mean ± SD of triplicates in a single experiment. * P < 0.05, ** P < 0.01, *** P < 0.001 for unpaired t -test. Data have been repeated in two ( e , f) or three ( a – d ) independent experiments.

    Techniques Used:

    a Knockdown of DAPK1 in HT-29 cells. HT-29 cells were transduced with pLL3.7-shCtrl or pLL3.7-shDAPK1, sorted, and then expressions of DAPK1, RIPK1 and RIPK3 were determined. b Increased zVAD+BV6-induced necroptosis in DAPK1-deficient HT-29 cells. Control and DAPK1-knockdown HT-29 cells were treated with zVAD (20 μM), BV6 (1 μM), Nec-1 (40 μM) as indicated for 24 h, cell death was quantitated by PI staining. Values are mean ± SD of triplicates in a single experiment. *** P < 0.001 for unpaired t -test. Results have been confirmed in three independent experiments. c Enhanced zVAD+BV6-induced phosphorylation of RIPK1, RIPK3 and MLKL in DAPK1-deficient HT-29 cells. Control and DAPK1-knockdown HT-29 cells were treated with zVAD+BV6 (0.5 μM, Z + B) and contents of RIPK1, pS166-RIPK1, RIPK3, pS227-RIPK3, MLKL, and pMLKL in cell lysates were determined at the indicated time points. Right panel, quantitation of pRIPK1(S166), pRIPK3 and pMLKL from three independent experiments using normalized intensity of pRIPK1(S166), pRIPK3 and pMLKL in DAPK1-knockdown HT29 cells at 9 h as 1. ** P < 0.01, *** P < 0.001 for two-way ANOVA followed by a Tukey’s multiple comparison test.
    Figure Legend Snippet: a Knockdown of DAPK1 in HT-29 cells. HT-29 cells were transduced with pLL3.7-shCtrl or pLL3.7-shDAPK1, sorted, and then expressions of DAPK1, RIPK1 and RIPK3 were determined. b Increased zVAD+BV6-induced necroptosis in DAPK1-deficient HT-29 cells. Control and DAPK1-knockdown HT-29 cells were treated with zVAD (20 μM), BV6 (1 μM), Nec-1 (40 μM) as indicated for 24 h, cell death was quantitated by PI staining. Values are mean ± SD of triplicates in a single experiment. *** P < 0.001 for unpaired t -test. Results have been confirmed in three independent experiments. c Enhanced zVAD+BV6-induced phosphorylation of RIPK1, RIPK3 and MLKL in DAPK1-deficient HT-29 cells. Control and DAPK1-knockdown HT-29 cells were treated with zVAD+BV6 (0.5 μM, Z + B) and contents of RIPK1, pS166-RIPK1, RIPK3, pS227-RIPK3, MLKL, and pMLKL in cell lysates were determined at the indicated time points. Right panel, quantitation of pRIPK1(S166), pRIPK3 and pMLKL from three independent experiments using normalized intensity of pRIPK1(S166), pRIPK3 and pMLKL in DAPK1-knockdown HT29 cells at 9 h as 1. ** P < 0.01, *** P < 0.001 for two-way ANOVA followed by a Tukey’s multiple comparison test.

    Techniques Used: Transduction, Staining, Quantitation Assay

    a , b Increased zVAD+AT-406-induced phosphorylation of RIPK1, RIPK3 and MLKL in Dapk1 −/− BMDMs. WT and Dapk1 −/− BMDMs were treated with zVAD (20 μM) plus AT-406 (0.6 μM, Z + A), and cell lysates were prepared at the indicated time points. The contents of RIPK1, pS166-RIPK1 ( a ), or RIPK3, pRIPK3, MLKL, and pMLKL ( b ) were determined by Western blot. Right panel, quantitation of pRIPK1(S166) ( a ) and pMLKL ( b ) from three independent experiments using normalized intensity of pRIPK1(S166) (3 h) and pMLKL (6 h) in Dapk1 −/− BMDMs as 1. *** P < 0.001 for two-way ANOVA followed by a Tukey’s multiple comparison test. c , d Increased activation of MLKL in Dapk1 −/− BMDMs stimulated by treatment with zVAD plus TNF or IFN-β. WT and Dapk1 −/− BMDMs were treated with zVAD in combination with TNF ( c ), or IFN-β ( d ) and levels of MLKL and pMLKL in cell lysates were determined at the indicated time points. Data are representative of three independent experiments.
    Figure Legend Snippet: a , b Increased zVAD+AT-406-induced phosphorylation of RIPK1, RIPK3 and MLKL in Dapk1 −/− BMDMs. WT and Dapk1 −/− BMDMs were treated with zVAD (20 μM) plus AT-406 (0.6 μM, Z + A), and cell lysates were prepared at the indicated time points. The contents of RIPK1, pS166-RIPK1 ( a ), or RIPK3, pRIPK3, MLKL, and pMLKL ( b ) were determined by Western blot. Right panel, quantitation of pRIPK1(S166) ( a ) and pMLKL ( b ) from three independent experiments using normalized intensity of pRIPK1(S166) (3 h) and pMLKL (6 h) in Dapk1 −/− BMDMs as 1. *** P < 0.001 for two-way ANOVA followed by a Tukey’s multiple comparison test. c , d Increased activation of MLKL in Dapk1 −/− BMDMs stimulated by treatment with zVAD plus TNF or IFN-β. WT and Dapk1 −/− BMDMs were treated with zVAD in combination with TNF ( c ), or IFN-β ( d ) and levels of MLKL and pMLKL in cell lysates were determined at the indicated time points. Data are representative of three independent experiments.

    Techniques Used: Western Blot, Quantitation Assay, Activation Assay

    a , b Enhanced zVAD+AT-406-induced binding of RIPK1 and RIPK3 to FADD/caspase-8 in Dapk1 −/− BMDMs. WT and Dapk1 −/− BMDMs were treated with zVAD+AT-406 (Z + A), and whole-cell lysates (WCL) were prepared at the indicated time points. WCL were immunoprecipitated with anti-FADD and contents of pRIPK1, RIPK1 and FADD in the precipitates and WCL were determined ( a ), or they were immunoprecipitated with anti-caspase-8 and the amounts of RIPK1, RIPK3, FADD and caspase-8 in the precipitates and WCL were determined ( b ). c , d Increased binding of RIPK1 and RIPK3 to FADD/caspase-8 in DAPK1-deficient HT-29 cells upon necroptotic induction. Control and DAPK1-knockdown HT-29 cells were treated with zVAD+BV6 + TNF (Z + B + T) ( c ) or zVAD+BV6 (Z + B) ( d ) before collecting WCL at the indicated time points. WCL were immunoprecipitated with anti-FADD and the contents of pRIPK1, RIPK1 and FADD in the precipitates and WCL were determined (c ), or we immunoprecipitated them with anti-caspse-8 and the amounts of RIPK1, RIPK3, FADD and caspase-8 were determined in the precipitates and WCL ( d ). Data are representative of three independent experiments.
    Figure Legend Snippet: a , b Enhanced zVAD+AT-406-induced binding of RIPK1 and RIPK3 to FADD/caspase-8 in Dapk1 −/− BMDMs. WT and Dapk1 −/− BMDMs were treated with zVAD+AT-406 (Z + A), and whole-cell lysates (WCL) were prepared at the indicated time points. WCL were immunoprecipitated with anti-FADD and contents of pRIPK1, RIPK1 and FADD in the precipitates and WCL were determined ( a ), or they were immunoprecipitated with anti-caspase-8 and the amounts of RIPK1, RIPK3, FADD and caspase-8 in the precipitates and WCL were determined ( b ). c , d Increased binding of RIPK1 and RIPK3 to FADD/caspase-8 in DAPK1-deficient HT-29 cells upon necroptotic induction. Control and DAPK1-knockdown HT-29 cells were treated with zVAD+BV6 + TNF (Z + B + T) ( c ) or zVAD+BV6 (Z + B) ( d ) before collecting WCL at the indicated time points. WCL were immunoprecipitated with anti-FADD and the contents of pRIPK1, RIPK1 and FADD in the precipitates and WCL were determined (c ), or we immunoprecipitated them with anti-caspse-8 and the amounts of RIPK1, RIPK3, FADD and caspase-8 were determined in the precipitates and WCL ( d ). Data are representative of three independent experiments.

    Techniques Used: Binding Assay, Immunoprecipitation

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    ProSci Incorporated anti mouse ripk3
    Anti Mouse 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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    mouse ripk3 cetsa  (ProSci Incorporated)
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    ( 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.
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    mouse ripk3  (ProSci Incorporated)
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    ProSci Incorporated mouse ripk3
    (A) Cultured <t>MCF7/TO-RIPK3</t> and HeLa/TO-RIPK3 cells were treated with DMSO or Dox(1μg/ml) induction for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells. The cell lysates were analyzed by western blotting using antibodies against RIPK3 or β-actin (lower panel). (B) Cultured MCF7/TO-RIPK3, KGN/TO-RIPK3, and HeLa/TO-RIPK3 cells were treated with DMSO, Dox, or Dox plus TSZ for 36 hours. Cell viability was determined by measuring cellular ATP levels. The data are represented as the mean ± SD of triplicate wells. (C) Cultured MCF7/TO-RIPK3 cells were treated with DMSO or Dox, plus the indicated agents for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells. The cell lysates were analyzed by western blotting using antibodies against RIPK3 or β-actin (lower panel). 20 μM Z, pan-caspase inhibitor z-VAD; 2 μM RIPA-56, RIPK1 inhibitor; 2 μM NSA, MLKL inhibitor. (D) Cultured MCF7 cells were infected with lentiviruses encoding RIPK3(WT), RIPK3(AAAA), RIPK3(K50A), and RIPK3(K50A)+GSK’872 plus Z for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells. *** P <0.001. The lysates were measured by western blotting using antibodies against RIPK3 or β-actin as indicated (lower panel). GSK’872, RIPK3 inhibitor. The online version of this article includes the following figure supplement(s) for : Figure supplement 1 . RIPK3-induced apoptosis in human granulosa lutein cells (KGN).
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    length mouse ripk3 isoform  (ProSci Incorporated)
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    ProSci Incorporated length mouse ripk3 isoform
    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.
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    ( 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.

    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

    ( 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.

    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

    ( 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.

    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

    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.

    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:

    ( 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.

    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:

    (A) Cultured MCF7/TO-RIPK3 and HeLa/TO-RIPK3 cells were treated with DMSO or Dox(1μg/ml) induction for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells. The cell lysates were analyzed by western blotting using antibodies against RIPK3 or β-actin (lower panel). (B) Cultured MCF7/TO-RIPK3, KGN/TO-RIPK3, and HeLa/TO-RIPK3 cells were treated with DMSO, Dox, or Dox plus TSZ for 36 hours. Cell viability was determined by measuring cellular ATP levels. The data are represented as the mean ± SD of triplicate wells. (C) Cultured MCF7/TO-RIPK3 cells were treated with DMSO or Dox, plus the indicated agents for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells. The cell lysates were analyzed by western blotting using antibodies against RIPK3 or β-actin (lower panel). 20 μM Z, pan-caspase inhibitor z-VAD; 2 μM RIPA-56, RIPK1 inhibitor; 2 μM NSA, MLKL inhibitor. (D) Cultured MCF7 cells were infected with lentiviruses encoding RIPK3(WT), RIPK3(AAAA), RIPK3(K50A), and RIPK3(K50A)+GSK’872 plus Z for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells. *** P <0.001. The lysates were measured by western blotting using antibodies against RIPK3 or β-actin as indicated (lower panel). GSK’872, RIPK3 inhibitor. The online version of this article includes the following figure supplement(s) for : Figure supplement 1 . RIPK3-induced apoptosis in human granulosa lutein cells (KGN).

    Journal: bioRxiv

    Article Title: A phosphorylation of RIPK3 kinase initiates an intracellular apoptotic pathway that promotes corpus luteum regression

    doi: 10.1101/2021.02.14.431152

    Figure Lengend Snippet: (A) Cultured MCF7/TO-RIPK3 and HeLa/TO-RIPK3 cells were treated with DMSO or Dox(1μg/ml) induction for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells. The cell lysates were analyzed by western blotting using antibodies against RIPK3 or β-actin (lower panel). (B) Cultured MCF7/TO-RIPK3, KGN/TO-RIPK3, and HeLa/TO-RIPK3 cells were treated with DMSO, Dox, or Dox plus TSZ for 36 hours. Cell viability was determined by measuring cellular ATP levels. The data are represented as the mean ± SD of triplicate wells. (C) Cultured MCF7/TO-RIPK3 cells were treated with DMSO or Dox, plus the indicated agents for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells. The cell lysates were analyzed by western blotting using antibodies against RIPK3 or β-actin (lower panel). 20 μM Z, pan-caspase inhibitor z-VAD; 2 μM RIPA-56, RIPK1 inhibitor; 2 μM NSA, MLKL inhibitor. (D) Cultured MCF7 cells were infected with lentiviruses encoding RIPK3(WT), RIPK3(AAAA), RIPK3(K50A), and RIPK3(K50A)+GSK’872 plus Z for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells. *** P <0.001. The lysates were measured by western blotting using antibodies against RIPK3 or β-actin as indicated (lower panel). GSK’872, RIPK3 inhibitor. The online version of this article includes the following figure supplement(s) for : Figure supplement 1 . RIPK3-induced apoptosis in human granulosa lutein cells (KGN).

    Article Snippet: Antibodies for mouse RIPK3 (#2283; WB, 1:1000; IHC, 1:100) were obtained from ProSci.

    Techniques: Cell Culture, Western Blot, Infection

    Cultured KGN/TO-RIPK3 cells were treated with DMSO or Dox(1μg/ml) induction for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells. The cell lysates were analyzed by western blotting using antibodies against RIPK3 or β-actin (lower panel).

    Journal: bioRxiv

    Article Title: A phosphorylation of RIPK3 kinase initiates an intracellular apoptotic pathway that promotes corpus luteum regression

    doi: 10.1101/2021.02.14.431152

    Figure Lengend Snippet: Cultured KGN/TO-RIPK3 cells were treated with DMSO or Dox(1μg/ml) induction for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells. The cell lysates were analyzed by western blotting using antibodies against RIPK3 or β-actin (lower panel).

    Article Snippet: Antibodies for mouse RIPK3 (#2283; WB, 1:1000; IHC, 1:100) were obtained from ProSci.

    Techniques: Cell Culture, Western Blot

    (A) Cultured MCF7/TO-RIPK3 cells were treated with DMSO or Dox plus the indicated agent for 24 hours. The cells were then harvested, and RIPK3 was immunoprecipitated from the cell lysates using anti-Flag resin. The cell lysates and immunocomplexes were analyzed by western blotting using antibodies as indicated. (B - E ) Cultured MCF7/TO-RIPK3(wild type (WT), RIPK1 -/- , Caspase8 -/- , FADD -/- , and cFLIF -/- ) cells were treated with DMSO or Dox induction for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells. The cell lysates were analyzed by western blotting using antibodies against RIPK1, Caspase8, FADD, cFLIP or β-actin (lower panel).

    Journal: bioRxiv

    Article Title: A phosphorylation of RIPK3 kinase initiates an intracellular apoptotic pathway that promotes corpus luteum regression

    doi: 10.1101/2021.02.14.431152

    Figure Lengend Snippet: (A) Cultured MCF7/TO-RIPK3 cells were treated with DMSO or Dox plus the indicated agent for 24 hours. The cells were then harvested, and RIPK3 was immunoprecipitated from the cell lysates using anti-Flag resin. The cell lysates and immunocomplexes were analyzed by western blotting using antibodies as indicated. (B - E ) Cultured MCF7/TO-RIPK3(wild type (WT), RIPK1 -/- , Caspase8 -/- , FADD -/- , and cFLIF -/- ) cells were treated with DMSO or Dox induction for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells. The cell lysates were analyzed by western blotting using antibodies against RIPK1, Caspase8, FADD, cFLIP or β-actin (lower panel).

    Article Snippet: Antibodies for mouse RIPK3 (#2283; WB, 1:1000; IHC, 1:100) were obtained from ProSci.

    Techniques: Cell Culture, Immunoprecipitation, Western Blot

    (A) Cultured MCF7/TO-RIPK3 and HeLa/TO-RIPK3 cells were treated with Dox plus z-VAD for 24 hours. RIPK3 was immunoprecipitated from the cell lysates using anti-Flag resin. The RIPK3 bands were excised and subjected to mass spectrometry analysis. RIPK3 specific phosphorylation site in MCF7/TO-RIPK3 cells is highlighted in red. (B) Cultured KGN/TO-RIPK3, MCF7/TO-RIPK3, and HeLa/TO-RIPK3 cells were treated with Dox plus z-VAD for 24 hours. The lysates were analyzed by western blotting using antibodies against the phopho-Serine164/theronine165 of RIPK3, Flag (RIPK3), and β-actin as indicated. (C) Cultured MCF7 stably transfected with either wild type RIPK3 (WT) or kinase-dead mutant (D160N) cells under the control of Dox-inducible promoter were treated with DMSO(-) or Dox plus z-VAD for 24 hours. The lysates were analyzed by western blotting using antibodies against the phopho-Serine164/theronine165 RIPK3, Flag (RIPK3), and β-actin as indicated. (D) Cultured 293T cells were transfected with Vector (Vec), RIPK3(WT), RIPK3(D160N), RIPK3(AAAA) (RIPK3-AAAA, residues 459-462 mutated to AAAA) and RIPK3(S164D/T165E) for 24 hours. The level of phospho-S227-RIPK3 and RIPK3 were measured by western blotting. (E and F) Cultured MCF7 (E) and KGN (F) cells were infected with lentiviruses encoding RIPK3(WT), RIPK3(S164D/T165E), RIPK3(S164A/T165A) and RIPK3(AAAA) plus z-VAD for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells. *** P <0.001. The lysates were measured by western blotting using antibodies against RIPK3 or β-actin as indicated (lower panel). (G) Cultured HeLa cells were infected with lentiviruses encoding RIPK3(WT), RIPK3(D160N), RIPK3(AAAA), and RIPK3(S164D/T165E) plus z-VAD for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells. ** P <0.01, *** P <0.001. The expressed RIPK3 in the cell lysates were measured by western blotting using antibodies against RIPK3 or β-actin as indicated (lower panel). Vector (Vec, control viruses) (H) Cultured HeLa cells were transfected with Flag-tagged RIPK3(WT), RIPK3(D160N), and RIPK3(S164D/T165E) for 24 hours. RIPK3 was immunoprecipitated using anti-Flag resin. The lysates and immunocomplexes were analyzed by western blotting using antibodies against RIPK1, Caspase-8, FADD, and RIPK3 as indicated. (I) Cultured MCF7/TO-RIPK3 and MCF7/TO-RIPK3(S164A/T165A) cells were treated with Dox plus Z for 24 hours. The cells were then harvested, and RIPK3 was immunoprecipitated from the cell lysates using anti-Flag resin. The cell lysates and immunocomplexes were analyzed by western blotting using antibodies as indicated. The online version of this article includes the following figure supplement(s) for figure 3: Figure supplement 1 . Characterization of RIPK3 auto-phosphorylation sites. Figure supplement 2 . The phosphorylation site of RIPK3 is conserved among different mammalian species.

    Journal: bioRxiv

    Article Title: A phosphorylation of RIPK3 kinase initiates an intracellular apoptotic pathway that promotes corpus luteum regression

    doi: 10.1101/2021.02.14.431152

    Figure Lengend Snippet: (A) Cultured MCF7/TO-RIPK3 and HeLa/TO-RIPK3 cells were treated with Dox plus z-VAD for 24 hours. RIPK3 was immunoprecipitated from the cell lysates using anti-Flag resin. The RIPK3 bands were excised and subjected to mass spectrometry analysis. RIPK3 specific phosphorylation site in MCF7/TO-RIPK3 cells is highlighted in red. (B) Cultured KGN/TO-RIPK3, MCF7/TO-RIPK3, and HeLa/TO-RIPK3 cells were treated with Dox plus z-VAD for 24 hours. The lysates were analyzed by western blotting using antibodies against the phopho-Serine164/theronine165 of RIPK3, Flag (RIPK3), and β-actin as indicated. (C) Cultured MCF7 stably transfected with either wild type RIPK3 (WT) or kinase-dead mutant (D160N) cells under the control of Dox-inducible promoter were treated with DMSO(-) or Dox plus z-VAD for 24 hours. The lysates were analyzed by western blotting using antibodies against the phopho-Serine164/theronine165 RIPK3, Flag (RIPK3), and β-actin as indicated. (D) Cultured 293T cells were transfected with Vector (Vec), RIPK3(WT), RIPK3(D160N), RIPK3(AAAA) (RIPK3-AAAA, residues 459-462 mutated to AAAA) and RIPK3(S164D/T165E) for 24 hours. The level of phospho-S227-RIPK3 and RIPK3 were measured by western blotting. (E and F) Cultured MCF7 (E) and KGN (F) cells were infected with lentiviruses encoding RIPK3(WT), RIPK3(S164D/T165E), RIPK3(S164A/T165A) and RIPK3(AAAA) plus z-VAD for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells. *** P <0.001. The lysates were measured by western blotting using antibodies against RIPK3 or β-actin as indicated (lower panel). (G) Cultured HeLa cells were infected with lentiviruses encoding RIPK3(WT), RIPK3(D160N), RIPK3(AAAA), and RIPK3(S164D/T165E) plus z-VAD for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells. ** P <0.01, *** P <0.001. The expressed RIPK3 in the cell lysates were measured by western blotting using antibodies against RIPK3 or β-actin as indicated (lower panel). Vector (Vec, control viruses) (H) Cultured HeLa cells were transfected with Flag-tagged RIPK3(WT), RIPK3(D160N), and RIPK3(S164D/T165E) for 24 hours. RIPK3 was immunoprecipitated using anti-Flag resin. The lysates and immunocomplexes were analyzed by western blotting using antibodies against RIPK1, Caspase-8, FADD, and RIPK3 as indicated. (I) Cultured MCF7/TO-RIPK3 and MCF7/TO-RIPK3(S164A/T165A) cells were treated with Dox plus Z for 24 hours. The cells were then harvested, and RIPK3 was immunoprecipitated from the cell lysates using anti-Flag resin. The cell lysates and immunocomplexes were analyzed by western blotting using antibodies as indicated. The online version of this article includes the following figure supplement(s) for figure 3: Figure supplement 1 . Characterization of RIPK3 auto-phosphorylation sites. Figure supplement 2 . The phosphorylation site of RIPK3 is conserved among different mammalian species.

    Article Snippet: Antibodies for mouse RIPK3 (#2283; WB, 1:1000; IHC, 1:100) were obtained from ProSci.

    Techniques: Cell Culture, Immunoprecipitation, Mass Spectrometry, Western Blot, Stable Transfection, Transfection, Mutagenesis, Plasmid Preparation, Infection

    (A) Cultured MCF7/TO-RIPK3 and MCF7/TO-RIPK3(S164A/T165A) cells were treated with DMSO (-) or Dox plus z-VAD for 24 hours. The lysates were analyzed by western blotting using antibodies against the phopho-Serine164/theronine165 of RIPK3, Flag (RIPK3), and β-actin as indicated. (B) Cultured KGN7/TO-RIPK3 and KGN/TO-RIPK3(S164A/T165A) cells were treated with DMSO or Dox plus z-VAD for 24 hours. The lysates were analyzed by western blotting using antibodies against the phopho-Serine164/theronine165 of RIPK3, Flag (RIPK3), and β-actin as indicated. ( C) Cultured MCF7/TO-RIPK3 and MCF7/TO-RIPK3(K50A) cells were treated with DMSO (-) or Dox plus z-VAD for 24 hours. The lysates were analyzed by western blotting using antibodies against the phopho-Serine164/theronine165 of RIPK3, Flag (RIPK3), and β-actin as indicated. (D) Cultured HeLa cells were infected with lentiviruses encoding wild type RIPK3(WT), and mutant RIPK3 including RIPK3(D160N), RIPK3(S164D/T165E), RIPK3(S164E), RIPK3(T165E), RIPK3(S164A/T165A), RIPK3(S164A), RIPK3(T165A) and RIPK3(AAAA) and treated with TSZ for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data represented as the mean ± SD of triplicate wells. ** P <0.01. The levels of expressed RIPK3 in the cell lysates were measured by western blotting (lower panel). (E) RIPK3 single site (S164E) mutation blocks auto-phosphorylation. 293T cells were transfected with Flag-tagged RIPK3(WT), RIPK3(D160N), RIPK3(S164D/T165E), RIPK3(S164E), RIPK3(T165E), RIPK3(S164A/T165A), RIPK3(S164A), RIPK3(T165A) and RIPK3(AAAA) for 24 hours. The level of p-S227-RIPK3 and RIPK3 were measured by western blotting. (F and G) Cultured MCF7 (E) and KGN (F) cells were infected with lentiviruses encoding wild type RIPK3(WT), and mutant forms of RIPK3(S164A/T165A), RIPK3(S164A) and RIPK3(T165A) and treated with z-VAD as indicated for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells. *** P <0.001. The levels of RIPK3 in the cell lysates were measured by western blotting (lower panel). (H) Cultured HeLa cells were transfected with Flag-tagged wild type RIPK3(WT), and mutant forms of RIPK3(T165E), RIPK3(S164D/T165E), and RIPK3(S164E) for 24 hours. RIPK3 was immunoprecipitated from the cell lysates using anti-Flag resin. The lysates and immunocomplexes were analyzed by western blotting using antibodies against RIPK1, caspase-8, RIPK3, and β-actin as indicated.

    Journal: bioRxiv

    Article Title: A phosphorylation of RIPK3 kinase initiates an intracellular apoptotic pathway that promotes corpus luteum regression

    doi: 10.1101/2021.02.14.431152

    Figure Lengend Snippet: (A) Cultured MCF7/TO-RIPK3 and MCF7/TO-RIPK3(S164A/T165A) cells were treated with DMSO (-) or Dox plus z-VAD for 24 hours. The lysates were analyzed by western blotting using antibodies against the phopho-Serine164/theronine165 of RIPK3, Flag (RIPK3), and β-actin as indicated. (B) Cultured KGN7/TO-RIPK3 and KGN/TO-RIPK3(S164A/T165A) cells were treated with DMSO or Dox plus z-VAD for 24 hours. The lysates were analyzed by western blotting using antibodies against the phopho-Serine164/theronine165 of RIPK3, Flag (RIPK3), and β-actin as indicated. ( C) Cultured MCF7/TO-RIPK3 and MCF7/TO-RIPK3(K50A) cells were treated with DMSO (-) or Dox plus z-VAD for 24 hours. The lysates were analyzed by western blotting using antibodies against the phopho-Serine164/theronine165 of RIPK3, Flag (RIPK3), and β-actin as indicated. (D) Cultured HeLa cells were infected with lentiviruses encoding wild type RIPK3(WT), and mutant RIPK3 including RIPK3(D160N), RIPK3(S164D/T165E), RIPK3(S164E), RIPK3(T165E), RIPK3(S164A/T165A), RIPK3(S164A), RIPK3(T165A) and RIPK3(AAAA) and treated with TSZ for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data represented as the mean ± SD of triplicate wells. ** P <0.01. The levels of expressed RIPK3 in the cell lysates were measured by western blotting (lower panel). (E) RIPK3 single site (S164E) mutation blocks auto-phosphorylation. 293T cells were transfected with Flag-tagged RIPK3(WT), RIPK3(D160N), RIPK3(S164D/T165E), RIPK3(S164E), RIPK3(T165E), RIPK3(S164A/T165A), RIPK3(S164A), RIPK3(T165A) and RIPK3(AAAA) for 24 hours. The level of p-S227-RIPK3 and RIPK3 were measured by western blotting. (F and G) Cultured MCF7 (E) and KGN (F) cells were infected with lentiviruses encoding wild type RIPK3(WT), and mutant forms of RIPK3(S164A/T165A), RIPK3(S164A) and RIPK3(T165A) and treated with z-VAD as indicated for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data are represented as the mean ± SD of triplicate wells. *** P <0.001. The levels of RIPK3 in the cell lysates were measured by western blotting (lower panel). (H) Cultured HeLa cells were transfected with Flag-tagged wild type RIPK3(WT), and mutant forms of RIPK3(T165E), RIPK3(S164D/T165E), and RIPK3(S164E) for 24 hours. RIPK3 was immunoprecipitated from the cell lysates using anti-Flag resin. The lysates and immunocomplexes were analyzed by western blotting using antibodies against RIPK1, caspase-8, RIPK3, and β-actin as indicated.

    Article Snippet: Antibodies for mouse RIPK3 (#2283; WB, 1:1000; IHC, 1:100) were obtained from ProSci.

    Techniques: Cell Culture, Western Blot, Infection, Mutagenesis, Transfection, Immunoprecipitation

    (A) The cell lysates from cultured HT29, HeLa, MCF7, and KGN cells were analyzed by western blotting using antibodies as indicated. (B and C) Cultured HeLa-RIPK3, MCF7/TO-RIPK3, and KGN/TO-RIPK3 cells were treated with the indicated stimuli for 36 hours. Cell viability was determined by measuring cellular ATP levels. The data represented as the mean ± SD of triplicate wells. 17AAG, Hsp90 inhibitor. (D and E) HeLa/TO-RIPK3 cells were treated with the indicated stimuli for 36 hours. Cell viability was determined by measuring cellular ATP levels in (D). The data represented as the mean ± SD of triplicate wells. *** P <0.001. 24 hours after treatment, the cell lysates were analyzed by western blotting using antibodies against phopho-Serine164/Theronine165 of RIPK3, RIPK3, and β-actin as indicated in (E). (F and G) Cultured MCF7/TO-RIPK3 cells co-transfected with HSP90 and CDC37 as indicated were treated with DMSO or Dox for 36 hours. Cell viability was determined by measuring cellular ATP levels in (F). The data are represented as the mean ± SD of triplicate wells. *** P <0.001. 24 hours after treatment, the cell lysates were analyzed by western blotting using antibodies against phopho-Serine164/Theronine165 of RIPK3, RIPK3, Hsp90, CDC37, and β-actin as indicated in (G). (H) Cultured HeLa/TO-RIPK3 cells were treated with Dox or Dox plus 17AAG for 24 hours. The cells were then harvested, and RIPK3 was immunoprecipitated from the cell lysates using anti-Flag resin. The cell lysates and immunocomplexes were analyzed by western blotting using antibodies as indicated. (I) Cultured HeLa/TO-RIPK3 cells were treated with Dox or Dox plus 17AAG for 24 hours. Immunofluorescence of the cells with Flag-RIPK3 (red) antibody. Counterstaining with DAPI (blue). Scale bar, 10 μm. Higher-power views (right panels) were acquired from the selected boxed areas from the left panel. (J) Cultured MCF7/TO-RIPK3 cells co-transfected with HSP90 and CDC37 as indicated were treated with Dox for 24 hours. Immunofluorescence of the cells with Flag-RIPK3 (red) antibody. Counterstaining with DAPI (blue). Scale bar, 10 μm. Higher-power views (right panels) were acquired from the selected boxed areas from the left panel. The online version of this article includes the following figure supplement(s) for figure 4: Figure supplement 1 . Hsp90/CDC37 chaperone determines the necroptotic or apoptotic function of RIPK3 kinase. Figure supplement 2 . RIPK3 form amyloid-like structure in MCF7 and KGN cells. Figure supplement 3 . Hsp90/CDC37 chaperone protein levels was low in corpus luteum and corpus albicans.

    Journal: bioRxiv

    Article Title: A phosphorylation of RIPK3 kinase initiates an intracellular apoptotic pathway that promotes corpus luteum regression

    doi: 10.1101/2021.02.14.431152

    Figure Lengend Snippet: (A) The cell lysates from cultured HT29, HeLa, MCF7, and KGN cells were analyzed by western blotting using antibodies as indicated. (B and C) Cultured HeLa-RIPK3, MCF7/TO-RIPK3, and KGN/TO-RIPK3 cells were treated with the indicated stimuli for 36 hours. Cell viability was determined by measuring cellular ATP levels. The data represented as the mean ± SD of triplicate wells. 17AAG, Hsp90 inhibitor. (D and E) HeLa/TO-RIPK3 cells were treated with the indicated stimuli for 36 hours. Cell viability was determined by measuring cellular ATP levels in (D). The data represented as the mean ± SD of triplicate wells. *** P <0.001. 24 hours after treatment, the cell lysates were analyzed by western blotting using antibodies against phopho-Serine164/Theronine165 of RIPK3, RIPK3, and β-actin as indicated in (E). (F and G) Cultured MCF7/TO-RIPK3 cells co-transfected with HSP90 and CDC37 as indicated were treated with DMSO or Dox for 36 hours. Cell viability was determined by measuring cellular ATP levels in (F). The data are represented as the mean ± SD of triplicate wells. *** P <0.001. 24 hours after treatment, the cell lysates were analyzed by western blotting using antibodies against phopho-Serine164/Theronine165 of RIPK3, RIPK3, Hsp90, CDC37, and β-actin as indicated in (G). (H) Cultured HeLa/TO-RIPK3 cells were treated with Dox or Dox plus 17AAG for 24 hours. The cells were then harvested, and RIPK3 was immunoprecipitated from the cell lysates using anti-Flag resin. The cell lysates and immunocomplexes were analyzed by western blotting using antibodies as indicated. (I) Cultured HeLa/TO-RIPK3 cells were treated with Dox or Dox plus 17AAG for 24 hours. Immunofluorescence of the cells with Flag-RIPK3 (red) antibody. Counterstaining with DAPI (blue). Scale bar, 10 μm. Higher-power views (right panels) were acquired from the selected boxed areas from the left panel. (J) Cultured MCF7/TO-RIPK3 cells co-transfected with HSP90 and CDC37 as indicated were treated with Dox for 24 hours. Immunofluorescence of the cells with Flag-RIPK3 (red) antibody. Counterstaining with DAPI (blue). Scale bar, 10 μm. Higher-power views (right panels) were acquired from the selected boxed areas from the left panel. The online version of this article includes the following figure supplement(s) for figure 4: Figure supplement 1 . Hsp90/CDC37 chaperone determines the necroptotic or apoptotic function of RIPK3 kinase. Figure supplement 2 . RIPK3 form amyloid-like structure in MCF7 and KGN cells. Figure supplement 3 . Hsp90/CDC37 chaperone protein levels was low in corpus luteum and corpus albicans.

    Article Snippet: Antibodies for mouse RIPK3 (#2283; WB, 1:1000; IHC, 1:100) were obtained from ProSci.

    Techniques: Cell Culture, Western Blot, Transfection, Immunoprecipitation, Immunofluorescence

    (A) HeLa/TO-RIPK3 and HeLa/TO-RIPK3-shRNA-HSP90 cells were treated with the indicated stimuli for 36 hours. Cell viability was determined by measuring cellular ATP levels. The data represented as the mean ± SD of triplicate wells. *** P <0.001. The cell lysates were analyzed by western blotting using antibodies as indicated (right panel). (B) L929 cells were treated with the indicated stimuli for 5 hours. Cell viability was determined by measuring cellular ATP levels. The data represented as the mean ± SD of triplicate wells. (C) L929( Ripk3 -/- )/TO-RIPK3 and L929( Ripk3 -/- )/TO-RIPK3-shRNA-HSP90 cells were treated with the indicated stimuli for 36 hours. Cell viability was determined by measuring cellular ATP levels. The data represented as the mean ± SD of triplicate wells. *** P < 0.001. The cell lysates were analyzed by western blotting using antibodies as indicated (right panel). (D and E ) L929( Ripk3 -/- )/TO-RIPK3 cells were treated with the indicated stimuli for 36 hours. Cell viability was determined by measuring cellular ATP levels in (D). The data represented as the mean ± SD of triplicate wells. ** P <0.01, *** P <0.001. 24 hours after treatment, the cell lysates were analyzed by western blotting using antibodies against phopho-Serine165/Theronine166 of RIPK3, RIPK3, and β-actin as indicated in (E).

    Journal: bioRxiv

    Article Title: A phosphorylation of RIPK3 kinase initiates an intracellular apoptotic pathway that promotes corpus luteum regression

    doi: 10.1101/2021.02.14.431152

    Figure Lengend Snippet: (A) HeLa/TO-RIPK3 and HeLa/TO-RIPK3-shRNA-HSP90 cells were treated with the indicated stimuli for 36 hours. Cell viability was determined by measuring cellular ATP levels. The data represented as the mean ± SD of triplicate wells. *** P <0.001. The cell lysates were analyzed by western blotting using antibodies as indicated (right panel). (B) L929 cells were treated with the indicated stimuli for 5 hours. Cell viability was determined by measuring cellular ATP levels. The data represented as the mean ± SD of triplicate wells. (C) L929( Ripk3 -/- )/TO-RIPK3 and L929( Ripk3 -/- )/TO-RIPK3-shRNA-HSP90 cells were treated with the indicated stimuli for 36 hours. Cell viability was determined by measuring cellular ATP levels. The data represented as the mean ± SD of triplicate wells. *** P < 0.001. The cell lysates were analyzed by western blotting using antibodies as indicated (right panel). (D and E ) L929( Ripk3 -/- )/TO-RIPK3 cells were treated with the indicated stimuli for 36 hours. Cell viability was determined by measuring cellular ATP levels in (D). The data represented as the mean ± SD of triplicate wells. ** P <0.01, *** P <0.001. 24 hours after treatment, the cell lysates were analyzed by western blotting using antibodies against phopho-Serine165/Theronine166 of RIPK3, RIPK3, and β-actin as indicated in (E).

    Article Snippet: Antibodies for mouse RIPK3 (#2283; WB, 1:1000; IHC, 1:100) were obtained from ProSci.

    Techniques: shRNA, Western Blot

    (A) Alignment of amino acid sequences of RIPK3 orthologs in five mammalian species. Amino acid residues conserved in 80% or more of the sequences are shaded in black. The putative phosphorylation residues are denoted by asterisks (*). (B) Cultured 293T cells were transfected with Vector (Vec), mouse RIPK3(WT), RIPK3(S165D/T166E), and RIPK3(S165A/T165A) for 24 hours. The level of phospho-S232-RIPK3 and RIPK3 were measured by western blotting. (C) Cultured mouse sarcoma cells L929( Ripk3 -/- ) were transfected with Vector, wild type mouse RIPK3, and mutant forms of mRIPK3(D161N), and mRIPK3(S165D/T166E) and treated with z-VAD or TSZ as indicated for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data represented as the mean ± SD of triplicate wells. The lysates were analyzed by western blotting using antibodies as indicated (lower panel).

    Journal: bioRxiv

    Article Title: A phosphorylation of RIPK3 kinase initiates an intracellular apoptotic pathway that promotes corpus luteum regression

    doi: 10.1101/2021.02.14.431152

    Figure Lengend Snippet: (A) Alignment of amino acid sequences of RIPK3 orthologs in five mammalian species. Amino acid residues conserved in 80% or more of the sequences are shaded in black. The putative phosphorylation residues are denoted by asterisks (*). (B) Cultured 293T cells were transfected with Vector (Vec), mouse RIPK3(WT), RIPK3(S165D/T166E), and RIPK3(S165A/T165A) for 24 hours. The level of phospho-S232-RIPK3 and RIPK3 were measured by western blotting. (C) Cultured mouse sarcoma cells L929( Ripk3 -/- ) were transfected with Vector, wild type mouse RIPK3, and mutant forms of mRIPK3(D161N), and mRIPK3(S165D/T166E) and treated with z-VAD or TSZ as indicated for 36 hours. Cell viability was determined by measuring cellular ATP levels (upper panel). The data represented as the mean ± SD of triplicate wells. The lysates were analyzed by western blotting using antibodies as indicated (lower panel).

    Article Snippet: Antibodies for mouse RIPK3 (#2283; WB, 1:1000; IHC, 1:100) were obtained from ProSci.

    Techniques: Cell Culture, Transfection, Plasmid Preparation, Western Blot, Mutagenesis

    (A) Cultured HeLa/TO-RIPK3 and MCF7/TO-RIPK3 cells were treated with DMSO or Dox plus TSZ for 24 hours. Immunofluorescence of the cells with Flag-RIPK3 (green) antibody. Counterstaining with DAPI (blue). Scale bar, 10 μm. Higher-power views (right panels) were acquired from the selected boxed areas from the left panel. (B) Cultured HT29 and KGN/TO-RIPK3 cells were treated with DMSO, TSZ, or Dox for 24 hours. Immunofluorescence of the cells with Flag-RIPK3 (green) antibody. Counterstaining with DAPI (blue). Scale bar, 10 μm. Higher-power views (right panels) were acquired from the selected boxed areas from the left panel. (C) Cultured HeLa/TO-RIPK3 and MCF7/TO-RIPK3 cells were treated with Dox plus Z for 24 hours. Immunofluorescence of the cells with Flag-RIPK3 (red) and p-S164/T165-RIPK3(green) antibody. Counterstaining with DAPI (blue). Scale bar, 10 μm. Higher-power views (right panels) were acquired from the selected boxed areas from the left panel. (D) Cultured MCF7/TO-RIPK3(WT, RIPK1 -/- , caspase-8 -/- and FADD -/- ) cells were treated with Dox plus Z for 24 hours. Immunofluorescence of the cells with Flag-RIPK3 (red) and p-S164/T165-RIPK3(green) antibody. Counterstaining with DAPI (blue). Scale bar, 10 μm. Higher-power views (right panels) were acquired from the selected boxed areas from the left panel. (E) Cultured MCF7/TO-RIPK3(WT, RIPK1 -/- , caspase-8 -/- and FADD -/- ) cells were treated with DMSO or Dox plus Z for 24 hours. The lysates were analyzed by western blotting using antibodies as indicated.

    Journal: bioRxiv

    Article Title: A phosphorylation of RIPK3 kinase initiates an intracellular apoptotic pathway that promotes corpus luteum regression

    doi: 10.1101/2021.02.14.431152

    Figure Lengend Snippet: (A) Cultured HeLa/TO-RIPK3 and MCF7/TO-RIPK3 cells were treated with DMSO or Dox plus TSZ for 24 hours. Immunofluorescence of the cells with Flag-RIPK3 (green) antibody. Counterstaining with DAPI (blue). Scale bar, 10 μm. Higher-power views (right panels) were acquired from the selected boxed areas from the left panel. (B) Cultured HT29 and KGN/TO-RIPK3 cells were treated with DMSO, TSZ, or Dox for 24 hours. Immunofluorescence of the cells with Flag-RIPK3 (green) antibody. Counterstaining with DAPI (blue). Scale bar, 10 μm. Higher-power views (right panels) were acquired from the selected boxed areas from the left panel. (C) Cultured HeLa/TO-RIPK3 and MCF7/TO-RIPK3 cells were treated with Dox plus Z for 24 hours. Immunofluorescence of the cells with Flag-RIPK3 (red) and p-S164/T165-RIPK3(green) antibody. Counterstaining with DAPI (blue). Scale bar, 10 μm. Higher-power views (right panels) were acquired from the selected boxed areas from the left panel. (D) Cultured MCF7/TO-RIPK3(WT, RIPK1 -/- , caspase-8 -/- and FADD -/- ) cells were treated with Dox plus Z for 24 hours. Immunofluorescence of the cells with Flag-RIPK3 (red) and p-S164/T165-RIPK3(green) antibody. Counterstaining with DAPI (blue). Scale bar, 10 μm. Higher-power views (right panels) were acquired from the selected boxed areas from the left panel. (E) Cultured MCF7/TO-RIPK3(WT, RIPK1 -/- , caspase-8 -/- and FADD -/- ) cells were treated with DMSO or Dox plus Z for 24 hours. The lysates were analyzed by western blotting using antibodies as indicated.

    Article Snippet: Antibodies for mouse RIPK3 (#2283; WB, 1:1000; IHC, 1:100) were obtained from ProSci.

    Techniques: Cell Culture, Immunofluorescence, Western Blot

    (A) Two guide RNA and donate oligo sequences of Ripk3(S165D/T166E) and Ripk3(S165A/T166A) knock-in mice. (B) Schematic of CRISPER-Cas9 strategy for the generation for Ripk3(S165D/T166E) and Ripk3(S165A/T166A) knock-in mice. The gene structure of RIPK3 and two guide RNA sequences targeting the exon 4 of RIPK3 were shown with the PAM sequences highlighted in red and blue. (C and D ) Macroscopic features (C) and body weights (D) of Ripk3 +/+ and Ripk3 ST-AA/ST-AA littermate mice at 14 days of age (n=10). The result from each individual animal was presented as an indicated dot. NS, not significant. (E and F ) Immunoblot of RIPK3 from lung extracts of 14 days old Ripk3 +/+ , Ripk3 ST-DE/ST-DE , and Ripk3 ST-AA/ST-AA littermates using antibodies against RIPK3 and GAPDH as indicated (n=3). (G) Histological analysis of brain, cerebellum, heart, kidney, and liver of Ripk3 +/+ and Ripk3 ST-DE/ST-DE littermate mice (n=5) at 14 days of age. Scale bar, 20 μm. PTC, Proximal tubular cell. (H) Cell viability measurement of bone marrow-derived macrophages from the Ripk3 +/+ and Ripk3 ST-AA/ST-AA littermate mice (n=3, 14 days) after treatment with the indicated necroptosis stimuli for 24 hours. Cell viability was determined by measuring cellular ATP levels. The data represented as the mean ± SD of triplicate wells.

    Journal: bioRxiv

    Article Title: A phosphorylation of RIPK3 kinase initiates an intracellular apoptotic pathway that promotes corpus luteum regression

    doi: 10.1101/2021.02.14.431152

    Figure Lengend Snippet: (A) Two guide RNA and donate oligo sequences of Ripk3(S165D/T166E) and Ripk3(S165A/T166A) knock-in mice. (B) Schematic of CRISPER-Cas9 strategy for the generation for Ripk3(S165D/T166E) and Ripk3(S165A/T166A) knock-in mice. The gene structure of RIPK3 and two guide RNA sequences targeting the exon 4 of RIPK3 were shown with the PAM sequences highlighted in red and blue. (C and D ) Macroscopic features (C) and body weights (D) of Ripk3 +/+ and Ripk3 ST-AA/ST-AA littermate mice at 14 days of age (n=10). The result from each individual animal was presented as an indicated dot. NS, not significant. (E and F ) Immunoblot of RIPK3 from lung extracts of 14 days old Ripk3 +/+ , Ripk3 ST-DE/ST-DE , and Ripk3 ST-AA/ST-AA littermates using antibodies against RIPK3 and GAPDH as indicated (n=3). (G) Histological analysis of brain, cerebellum, heart, kidney, and liver of Ripk3 +/+ and Ripk3 ST-DE/ST-DE littermate mice (n=5) at 14 days of age. Scale bar, 20 μm. PTC, Proximal tubular cell. (H) Cell viability measurement of bone marrow-derived macrophages from the Ripk3 +/+ and Ripk3 ST-AA/ST-AA littermate mice (n=3, 14 days) after treatment with the indicated necroptosis stimuli for 24 hours. Cell viability was determined by measuring cellular ATP levels. The data represented as the mean ± SD of triplicate wells.

    Article Snippet: Antibodies for mouse RIPK3 (#2283; WB, 1:1000; IHC, 1:100) were obtained from ProSci.

    Techniques: Knock-In, Western Blot, Derivative Assay

    (A and B ) Macroscopic features (A) and body weights (B) of Ripk3 +/+ and Ripk3 ST-DE/ST-DE littermate mice at 14 days of age (n≥9). The result from each individual animal is presented as an indicated dot. *** P <0.001. (C) Kaplan-Meier plot of survival of Ripk3 +/+ and Ripk3 ST-DE/ST-DE littermate mice (n=10 for each genotype) after birth within two months. *** P <0.001. (D) Histological analysis of large intestine, small intestine, lung, and spleen of Ripk3 +/+ and Ripk3 ST-DE/ST-DE littermate mice (n=5) at 14 days of age. Scale bar, 20 μm. (E and F) Representative immunohistochemistry (IHC) images of the large intestine, small intestine, lung, and spleen of Ripk3 +/+ and Ripk3 ST-DE/ST-DE littermate mice (n=5, 14 days) stained with a Cleaved-Caspase3 (C-C3) antibody in (E). C-C3 positive cells were counted in two fields per organ and quantified in (F). Scale bar, 10 μm. Data represent the mean ± s.e.m. ** P <0.01, *** P <0.001. (G) Cell viability measurement of bone marrow-derived macrophages from the Ripk3 +/+ and Ripk3 ST-DE/ST-DE littermate mice (n=3, 14 days) after treatment with the indicated Z-VAD or necroptosis stimuli for 24 hours. Cell viability was determined by measuring cellular ATP levels. The data are represented as the mean ± SD of triplicate wells. The online version of this article includes the following figure supplement(s) for figure 5: Figure supplement 1 . Generation of Ripk3 ST-DE/ST-DE and Ripk3 ST-AA/ST-AA mice.

    Journal: bioRxiv

    Article Title: A phosphorylation of RIPK3 kinase initiates an intracellular apoptotic pathway that promotes corpus luteum regression

    doi: 10.1101/2021.02.14.431152

    Figure Lengend Snippet: (A and B ) Macroscopic features (A) and body weights (B) of Ripk3 +/+ and Ripk3 ST-DE/ST-DE littermate mice at 14 days of age (n≥9). The result from each individual animal is presented as an indicated dot. *** P <0.001. (C) Kaplan-Meier plot of survival of Ripk3 +/+ and Ripk3 ST-DE/ST-DE littermate mice (n=10 for each genotype) after birth within two months. *** P <0.001. (D) Histological analysis of large intestine, small intestine, lung, and spleen of Ripk3 +/+ and Ripk3 ST-DE/ST-DE littermate mice (n=5) at 14 days of age. Scale bar, 20 μm. (E and F) Representative immunohistochemistry (IHC) images of the large intestine, small intestine, lung, and spleen of Ripk3 +/+ and Ripk3 ST-DE/ST-DE littermate mice (n=5, 14 days) stained with a Cleaved-Caspase3 (C-C3) antibody in (E). C-C3 positive cells were counted in two fields per organ and quantified in (F). Scale bar, 10 μm. Data represent the mean ± s.e.m. ** P <0.01, *** P <0.001. (G) Cell viability measurement of bone marrow-derived macrophages from the Ripk3 +/+ and Ripk3 ST-DE/ST-DE littermate mice (n=3, 14 days) after treatment with the indicated Z-VAD or necroptosis stimuli for 24 hours. Cell viability was determined by measuring cellular ATP levels. The data are represented as the mean ± SD of triplicate wells. The online version of this article includes the following figure supplement(s) for figure 5: Figure supplement 1 . Generation of Ripk3 ST-DE/ST-DE and Ripk3 ST-AA/ST-AA mice.

    Article Snippet: Antibodies for mouse RIPK3 (#2283; WB, 1:1000; IHC, 1:100) were obtained from ProSci.

    Techniques: Immunohistochemistry, Staining, Derivative Assay

    (A) Western blot analysis of RIPK1, RIPK3, and MLKL levels in perfused mouse ovary extracts of different ages. Each group is representative of at least 3 mice. (B) H&E and immunofluorescence (IF) imaging of an 8-month-old ovary. Two adjacent sections were analyzed. One section was stained with H&E, and the other was IF stained with a RIPK3 antibody (red) and DAPI (blue). Scale bar, 500 μm. Higher-power views of selected areas were acquired in a (primordia follicle), b (secondary follicle), c (corpus luteum), and d (corpus albicans) as indicated. PF, primary follicle; CL, corpus luteum; CA, corpus albicans. (C) Ovarian PGF 2α levels of wild-type mice (n=8) at the indicated age assayed by ELISA. Data represent the mean ± s.e.m. ** P <0.01, *** P <0.001. (D) Immunofluorescence images of a RIPK3 C-terminus HA-3×Flag knock-in mouse ovary (n=5; 12 months) stained with antibodies against prostaglandin F receptor (PTGFR, green) and Flag (red). Counterstaining with DAPI (blue). Scale bar, 500 μm. Higher-power views (right panels) were acquired from the indicated boxed area in the second lower left panel. CL, corpus luteum. CA, corpus albicans. Scale bar, 100 μm. (E) Western blot analysis of p-S164/T165-RIPK3 and RIPK3 levels in extracts from perfused ovaries prepared from mice at the indicated age. Each group is representative of at least 3 mice. (F) Immunofluorescence images of ovaries from Ripk3 +/+ and Ripk3 -/- mice (4 Month, 8 Month and 12 Month; n=3) at the indicated ages stained with the p-S164/T165-RIPK3 antibody (red). Counterstaining with DAPI (blue). Scale bar, 200 μm. Higher-power views (lower two panels) were acquired from the selected boxed areas from the upper panel. (CL), b (CL), c (CL, CA) and d (CL). F, follicle; CL, corpus luteum; CA, corpus albicans. Scale bar, 100 μm. The online version of this article includes the following figure supplement(s) for figure 6: Figure supplement 1 . Generation of RIPK3 C-terminus HA-3×Flag knock-in and Ptgfr -/- mice.

    Journal: bioRxiv

    Article Title: A phosphorylation of RIPK3 kinase initiates an intracellular apoptotic pathway that promotes corpus luteum regression

    doi: 10.1101/2021.02.14.431152

    Figure Lengend Snippet: (A) Western blot analysis of RIPK1, RIPK3, and MLKL levels in perfused mouse ovary extracts of different ages. Each group is representative of at least 3 mice. (B) H&E and immunofluorescence (IF) imaging of an 8-month-old ovary. Two adjacent sections were analyzed. One section was stained with H&E, and the other was IF stained with a RIPK3 antibody (red) and DAPI (blue). Scale bar, 500 μm. Higher-power views of selected areas were acquired in a (primordia follicle), b (secondary follicle), c (corpus luteum), and d (corpus albicans) as indicated. PF, primary follicle; CL, corpus luteum; CA, corpus albicans. (C) Ovarian PGF 2α levels of wild-type mice (n=8) at the indicated age assayed by ELISA. Data represent the mean ± s.e.m. ** P <0.01, *** P <0.001. (D) Immunofluorescence images of a RIPK3 C-terminus HA-3×Flag knock-in mouse ovary (n=5; 12 months) stained with antibodies against prostaglandin F receptor (PTGFR, green) and Flag (red). Counterstaining with DAPI (blue). Scale bar, 500 μm. Higher-power views (right panels) were acquired from the indicated boxed area in the second lower left panel. CL, corpus luteum. CA, corpus albicans. Scale bar, 100 μm. (E) Western blot analysis of p-S164/T165-RIPK3 and RIPK3 levels in extracts from perfused ovaries prepared from mice at the indicated age. Each group is representative of at least 3 mice. (F) Immunofluorescence images of ovaries from Ripk3 +/+ and Ripk3 -/- mice (4 Month, 8 Month and 12 Month; n=3) at the indicated ages stained with the p-S164/T165-RIPK3 antibody (red). Counterstaining with DAPI (blue). Scale bar, 200 μm. Higher-power views (lower two panels) were acquired from the selected boxed areas from the upper panel. (CL), b (CL), c (CL, CA) and d (CL). F, follicle; CL, corpus luteum; CA, corpus albicans. Scale bar, 100 μm. The online version of this article includes the following figure supplement(s) for figure 6: Figure supplement 1 . Generation of RIPK3 C-terminus HA-3×Flag knock-in and Ptgfr -/- mice.

    Article Snippet: Antibodies for mouse RIPK3 (#2283; WB, 1:1000; IHC, 1:100) were obtained from ProSci.

    Techniques: Western Blot, Immunofluorescence, Imaging, Staining, Enzyme-linked Immunosorbent Assay, Knock-In

    (A) Schematic of CRISPER-Cas9 strategy for RIPK3 C-terminus HA-3×Flag knock-in mice. The gene structure of RIPK3 and guide RNA sequences targeting the Ripk3 were shown with the PAM sequences highlighted in red. (B) Western blotting analysis using protein extracts from the ovary of wild type, heterozygous knock-in, and homozygous knock-in mice generated as illustrated in (A). (C) Schematic of CRISPER-Cas9 strategy for the generation for Ptgfr -/- mice. The gene structure of PTGFR and two guide RNA sequences targeting the Ptgfr were shown with the PAM sequences highlighted in red. (D) Immunoblot of PTGFR from ovary extracts of 2-month old Ptgfr +/+ and Ptgfr -/- littermates using antibodies against PTGFR and GAPDH as indicated (n=3).

    Journal: bioRxiv

    Article Title: A phosphorylation of RIPK3 kinase initiates an intracellular apoptotic pathway that promotes corpus luteum regression

    doi: 10.1101/2021.02.14.431152

    Figure Lengend Snippet: (A) Schematic of CRISPER-Cas9 strategy for RIPK3 C-terminus HA-3×Flag knock-in mice. The gene structure of RIPK3 and guide RNA sequences targeting the Ripk3 were shown with the PAM sequences highlighted in red. (B) Western blotting analysis using protein extracts from the ovary of wild type, heterozygous knock-in, and homozygous knock-in mice generated as illustrated in (A). (C) Schematic of CRISPER-Cas9 strategy for the generation for Ptgfr -/- mice. The gene structure of PTGFR and two guide RNA sequences targeting the Ptgfr were shown with the PAM sequences highlighted in red. (D) Immunoblot of PTGFR from ovary extracts of 2-month old Ptgfr +/+ and Ptgfr -/- littermates using antibodies against PTGFR and GAPDH as indicated (n=3).

    Article Snippet: Antibodies for mouse RIPK3 (#2283; WB, 1:1000; IHC, 1:100) were obtained from ProSci.

    Techniques: Knock-In, Western Blot, Generated

    (A and B ) Immunofluorescence of ovary from wild type mice (8 Month; n=3) with RIPK3 (red) and HSP90 (green) antibody in (A). Higher-power views of selected areas were acquired in right panel. The HSP90/RIPK3 levels were quantified in (B). F, follicle. CA, corpus albicans. Scale bar, 100/200 μm. ( C and D ) Immunofluorescence of ovary from wild type mice (8 Month; n=3) with RIPK3 (red) and CDC37 (green) antibody in (C). Higher-power views of selected areas were acquired in right panel. The CDC37/RIPK3 levels were quantified in (D). F, follicle. CA, corpus albicans. Scale bar, 200 μm.

    Journal: bioRxiv

    Article Title: A phosphorylation of RIPK3 kinase initiates an intracellular apoptotic pathway that promotes corpus luteum regression

    doi: 10.1101/2021.02.14.431152

    Figure Lengend Snippet: (A and B ) Immunofluorescence of ovary from wild type mice (8 Month; n=3) with RIPK3 (red) and HSP90 (green) antibody in (A). Higher-power views of selected areas were acquired in right panel. The HSP90/RIPK3 levels were quantified in (B). F, follicle. CA, corpus albicans. Scale bar, 100/200 μm. ( C and D ) Immunofluorescence of ovary from wild type mice (8 Month; n=3) with RIPK3 (red) and CDC37 (green) antibody in (C). Higher-power views of selected areas were acquired in right panel. The CDC37/RIPK3 levels were quantified in (D). F, follicle. CA, corpus albicans. Scale bar, 200 μm.

    Article Snippet: Antibodies for mouse RIPK3 (#2283; WB, 1:1000; IHC, 1:100) were obtained from ProSci.

    Techniques: Immunofluorescence

    (A to C) Primary granulosal lutein cells (WT, Ripk3 -/- ) were isolated from 3-month-old mice ovaries. The cells were treated with Dinoprost Tromethamine (DT) at the indicated concentration for 36 hours in (A); with 1.5 μM DT at the indicated time in (B); or with 1.5 μM DT for 36 hours in (C). The cell lysates from the DT-treated cells were analyzed by western blotting using antibodies as indicated. (D and E) Ptgfr +/+ and Ptgfr -/- littermate female mice (n=16; 25-26 days) were given 7.5 IU pregnant mare serum gonadotropin (PMSG) intraperitoneally(IP) followed by 7.5 IU serum gonadotropin and chorionic gonadotropin (SCG) 46 hours later to synchronize ovulation. The animals were then injected with Dinoprost Tromethamine (DT) (10 micrograms, i.p.) or saline 24 hours post-ovulation. Ovaries were then collected 12 hours later and stained with anti-RIPK3 antibody (red) in (D). The ovary lysates were analyzed by western blotting using antibodies as indicated in (E). (*) indicates Corpus luteum. Counterstaining with DAPI (blue). Scale bar, 500 μm. (F and G) wild type(WT), Ripk3 -/- , Ripk3 S165A-T166A/S165A-T166A , Fadd -/- Mlkl -/- and Ptgfr -/- female mice (each group, n=16; 25-26 days) were treated as in (D and F). Ovaries from each group were then collected 24 hours after injecting with DT and stained with anti-cleaved-caspase3 antibody in (F). The Cleaved-Caspase3 + cells were counted in five fields per ovary Corpus luteum(CL) and quantified in (G). Scale bar, 20 μm. Data represent the mean ± s.e.m. ** P <0.01, *** P <0.001. (H) wild type female mice (n=3; 25-26 days) were treated as in (D and F). Ovaries were then collected 12 hours after injecting with DT and stained with anti-cleaved-caspase3(red) and p-S164/T165-RIPK3(green) antibody. Counterstaining with DAPI (blue). Scale bar, 100 μm. The online version of this article includes the following figure supplement(s) for figure 7: Figure supplement 1 . Prostaglandin F2alpha (PGF 2α ) stimulates RIPK3 expression through the MAPK pathway.

    Journal: bioRxiv

    Article Title: A phosphorylation of RIPK3 kinase initiates an intracellular apoptotic pathway that promotes corpus luteum regression

    doi: 10.1101/2021.02.14.431152

    Figure Lengend Snippet: (A to C) Primary granulosal lutein cells (WT, Ripk3 -/- ) were isolated from 3-month-old mice ovaries. The cells were treated with Dinoprost Tromethamine (DT) at the indicated concentration for 36 hours in (A); with 1.5 μM DT at the indicated time in (B); or with 1.5 μM DT for 36 hours in (C). The cell lysates from the DT-treated cells were analyzed by western blotting using antibodies as indicated. (D and E) Ptgfr +/+ and Ptgfr -/- littermate female mice (n=16; 25-26 days) were given 7.5 IU pregnant mare serum gonadotropin (PMSG) intraperitoneally(IP) followed by 7.5 IU serum gonadotropin and chorionic gonadotropin (SCG) 46 hours later to synchronize ovulation. The animals were then injected with Dinoprost Tromethamine (DT) (10 micrograms, i.p.) or saline 24 hours post-ovulation. Ovaries were then collected 12 hours later and stained with anti-RIPK3 antibody (red) in (D). The ovary lysates were analyzed by western blotting using antibodies as indicated in (E). (*) indicates Corpus luteum. Counterstaining with DAPI (blue). Scale bar, 500 μm. (F and G) wild type(WT), Ripk3 -/- , Ripk3 S165A-T166A/S165A-T166A , Fadd -/- Mlkl -/- and Ptgfr -/- female mice (each group, n=16; 25-26 days) were treated as in (D and F). Ovaries from each group were then collected 24 hours after injecting with DT and stained with anti-cleaved-caspase3 antibody in (F). The Cleaved-Caspase3 + cells were counted in five fields per ovary Corpus luteum(CL) and quantified in (G). Scale bar, 20 μm. Data represent the mean ± s.e.m. ** P <0.01, *** P <0.001. (H) wild type female mice (n=3; 25-26 days) were treated as in (D and F). Ovaries were then collected 12 hours after injecting with DT and stained with anti-cleaved-caspase3(red) and p-S164/T165-RIPK3(green) antibody. Counterstaining with DAPI (blue). Scale bar, 100 μm. The online version of this article includes the following figure supplement(s) for figure 7: Figure supplement 1 . Prostaglandin F2alpha (PGF 2α ) stimulates RIPK3 expression through the MAPK pathway.

    Article Snippet: Antibodies for mouse RIPK3 (#2283; WB, 1:1000; IHC, 1:100) were obtained from ProSci.

    Techniques: Isolation, Concentration Assay, Western Blot, Injection, Staining, Expressing

    (A) Primary granulosal lutein cells were isolated from the 3-month-old mice ovary. The cells were then treated with 1 μM DT or plus MAPK inhibitors PD-98059 (5 μM) and U0126 (5 μM) as indicated for 36 hours. The lysates were analyzed by western blotting using antibodies as indicated. (B) Primary granulosal lutein cells were isolated from 3-month-old mice ovaries. The cells were treated with 1 μM DT at the indicated time. The cell lysates from the DT-treated cells were analyzed by western blotting using antibodies as indicated. (C) Diagram of induction of corpus luteum regression in vivo . (D) Ripk3 +/+ and Ripk3 S165A-T166A/S165A-T166A and littermate female mice (n=3; 25-26 days) were given 7.5 IU pregnant mare serum gonadotropin (PMSG) intraperitoneally(IP) followed by 7.5 IU serum gonadotropin and chorionic gonadotropin (SCG) 46 hours later to synchronize ovulation. The animals were then injected with Dinoprost Tromethamine (DT) (10 micrograms, i.p.) or saline 24 hours post-ovulation. The ovary lysates were analyzed by western blotting using antibodies as indicated.

    Journal: bioRxiv

    Article Title: A phosphorylation of RIPK3 kinase initiates an intracellular apoptotic pathway that promotes corpus luteum regression

    doi: 10.1101/2021.02.14.431152

    Figure Lengend Snippet: (A) Primary granulosal lutein cells were isolated from the 3-month-old mice ovary. The cells were then treated with 1 μM DT or plus MAPK inhibitors PD-98059 (5 μM) and U0126 (5 μM) as indicated for 36 hours. The lysates were analyzed by western blotting using antibodies as indicated. (B) Primary granulosal lutein cells were isolated from 3-month-old mice ovaries. The cells were treated with 1 μM DT at the indicated time. The cell lysates from the DT-treated cells were analyzed by western blotting using antibodies as indicated. (C) Diagram of induction of corpus luteum regression in vivo . (D) Ripk3 +/+ and Ripk3 S165A-T166A/S165A-T166A and littermate female mice (n=3; 25-26 days) were given 7.5 IU pregnant mare serum gonadotropin (PMSG) intraperitoneally(IP) followed by 7.5 IU serum gonadotropin and chorionic gonadotropin (SCG) 46 hours later to synchronize ovulation. The animals were then injected with Dinoprost Tromethamine (DT) (10 micrograms, i.p.) or saline 24 hours post-ovulation. The ovary lysates were analyzed by western blotting using antibodies as indicated.

    Article Snippet: Antibodies for mouse RIPK3 (#2283; WB, 1:1000; IHC, 1:100) were obtained from ProSci.

    Techniques: Isolation, Western Blot, In Vivo, Injection

    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.

    Journal: bioRxiv

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

    doi: 10.1101/2020.10.26.356063

    Figure Lengend 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.

    Article Snippet: These signals are likely to be spliced isoforms of mouse RIPK3 of varying lengths, each of which harbour the kinase domain antigen (residues 2-353), which are therefore not detected by antibodies directed towards the C-terminus of the full-length mouse RIPK3 isoform, such as ProSci 2283.

    Techniques: 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.

    Journal: bioRxiv

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

    doi: 10.1101/2020.10.26.356063

    Figure Lengend 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.

    Article Snippet: These signals are likely to be spliced isoforms of mouse RIPK3 of varying lengths, each of which harbour the kinase domain antigen (residues 2-353), which are therefore not detected by antibodies directed towards the C-terminus of the full-length mouse RIPK3 isoform, such as ProSci 2283.

    Techniques: Quantitation Assay, Produced, Staining, Western Blot