anti phospho mouse ripk3 ser232  (Danaher Inc)


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    Danaher Inc anti phospho mouse ripk3 ser232
    Anti Phospho Mouse Ripk3 Ser232, supplied by Danaher Inc, 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/anti phospho mouse ripk3 ser232/product/Danaher Inc
    Average 86 stars, based on 1 article reviews
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    anti phospho mouse ripk3 ser232 - by Bioz Stars, 2024-09
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    anti mouse p ripk3  (Cell Signaling Technology Inc)


    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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    Cell Signaling Technology Inc anti mouse p ripk3
    Genetic loss of <t>RIPK3</t> leads to hyperproliferation of DP thymocytes contributing to thymic lymphoma. A) Kaplan–Meier survival curve (Long‐rank test) in Ripk3 +/+ ( n = 51) and Ripk3 −/− ( n = 40) mice. B) Spontaneous tumors found in various organs and their frequency in Ripk3 +/+ and Ripk3 −/− animals. C) Thymus from Ripk3 +/+ and Ripk3 −/− was shown (left panel) and its weight was shown in the graph (right panel). ( n = 13 for each group). D) Representative H&E images of thymus from both Ripk3 +/+ and Ripk3 −/− mice. E) Percentage of the thymic T cell subsets (left panel) and the absolute cell number (right panel) by the indicated thymic subsets are shown in each graph. ( n = 7 for each group). F) Relative fluorescence of Ki‐67 in CD4 + CD8 + double positive (DP) T cells under anti‐CD3/CD28 stimulation. Total thymocytes from Ripk3 +/+ ( n = 5) and Ripk3 −/− ( n = 5) mice were stimulated with anti‐CD3 (1 µg mL −1 ) and anti‐CD28 (2 µg mL −1 ) or without, and then Ki‐67 in DP cell was measured by FACS. Statistical analyses were performed using the two‐tailed unpaired Student t ‐test. P values below 0.05 were considered significant in the following manner: * p < 0.05, ** p < 0.01, *** p < 0.001.
    Anti Mouse P Ripk3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti mouse p ripk3/product/Cell Signaling Technology Inc
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti mouse p ripk3 - by Bioz Stars, 2024-09
    95/100 stars

    Images

    1) Product Images from "LCK‐Mediated RIPK3 Activation Controls Double‐Positive Thymocyte Proliferation and Restrains Thymic Lymphoma by Regulating the PP2A‐ERK Axis"

    Article Title: LCK‐Mediated RIPK3 Activation Controls Double‐Positive Thymocyte Proliferation and Restrains Thymic Lymphoma by Regulating the PP2A‐ERK Axis

    Journal: Advanced Science

    doi: 10.1002/advs.202204522

    Genetic loss of RIPK3 leads to hyperproliferation of DP thymocytes contributing to thymic lymphoma. A) Kaplan–Meier survival curve (Long‐rank test) in Ripk3 +/+ ( n = 51) and Ripk3 −/− ( n = 40) mice. B) Spontaneous tumors found in various organs and their frequency in Ripk3 +/+ and Ripk3 −/− animals. C) Thymus from Ripk3 +/+ and Ripk3 −/− was shown (left panel) and its weight was shown in the graph (right panel). ( n = 13 for each group). D) Representative H&E images of thymus from both Ripk3 +/+ and Ripk3 −/− mice. E) Percentage of the thymic T cell subsets (left panel) and the absolute cell number (right panel) by the indicated thymic subsets are shown in each graph. ( n = 7 for each group). F) Relative fluorescence of Ki‐67 in CD4 + CD8 + double positive (DP) T cells under anti‐CD3/CD28 stimulation. Total thymocytes from Ripk3 +/+ ( n = 5) and Ripk3 −/− ( n = 5) mice were stimulated with anti‐CD3 (1 µg mL −1 ) and anti‐CD28 (2 µg mL −1 ) or without, and then Ki‐67 in DP cell was measured by FACS. Statistical analyses were performed using the two‐tailed unpaired Student t ‐test. P values below 0.05 were considered significant in the following manner: * p < 0.05, ** p < 0.01, *** p < 0.001.
    Figure Legend Snippet: Genetic loss of RIPK3 leads to hyperproliferation of DP thymocytes contributing to thymic lymphoma. A) Kaplan–Meier survival curve (Long‐rank test) in Ripk3 +/+ ( n = 51) and Ripk3 −/− ( n = 40) mice. B) Spontaneous tumors found in various organs and their frequency in Ripk3 +/+ and Ripk3 −/− animals. C) Thymus from Ripk3 +/+ and Ripk3 −/− was shown (left panel) and its weight was shown in the graph (right panel). ( n = 13 for each group). D) Representative H&E images of thymus from both Ripk3 +/+ and Ripk3 −/− mice. E) Percentage of the thymic T cell subsets (left panel) and the absolute cell number (right panel) by the indicated thymic subsets are shown in each graph. ( n = 7 for each group). F) Relative fluorescence of Ki‐67 in CD4 + CD8 + double positive (DP) T cells under anti‐CD3/CD28 stimulation. Total thymocytes from Ripk3 +/+ ( n = 5) and Ripk3 −/− ( n = 5) mice were stimulated with anti‐CD3 (1 µg mL −1 ) and anti‐CD28 (2 µg mL −1 ) or without, and then Ki‐67 in DP cell was measured by FACS. Statistical analyses were performed using the two‐tailed unpaired Student t ‐test. P values below 0.05 were considered significant in the following manner: * p < 0.05, ** p < 0.01, *** p < 0.001.

    Techniques Used: Fluorescence, Two Tailed Test

    RIPK3‐MLKL axis does not impact the proliferation and death of DP thymocytes. A) Total thymocytes from Ripk3 +/+ ( n = 6) and Ripk3 −/− ( n = 6) mice were treated with TNF‐ α (50 ng mL −1 ), z‐VAD (20 µ m ) and Smac mimetic (200 n m ) for 5 and 24 h. Dead cells were identified by Annexin V and PI staining. B) The expression level of MLKL protein in primary immune cells. MLKL expression was measured by Western blot analysis. C) Representative histograms were the MLKL expression of the indicated thymic and splenic subsets. Expression levels of MLKL were measured by flow cytometry. Total thymocytes and splenocytes were treated with TNF‐ α (50 ng mL −1 ), z‐VAD (20 µ m ) and Smac mimetic (200 n m ) for the indicated time or without, and cells were analyzed by Annexin V and Propidium Iodide (PI) staining and measured by D) Flow Cytometry. RIPK3, MLKL and p ‐MLKL protein was measured by E) Western blot analysis. Statistical analyses were performed using the two‐tailed unpaired Student t ‐test. P values below 0.05 were considered significant in the following manner: * p < 0.05, ** p < 0.01, *** p < 0.001.
    Figure Legend Snippet: RIPK3‐MLKL axis does not impact the proliferation and death of DP thymocytes. A) Total thymocytes from Ripk3 +/+ ( n = 6) and Ripk3 −/− ( n = 6) mice were treated with TNF‐ α (50 ng mL −1 ), z‐VAD (20 µ m ) and Smac mimetic (200 n m ) for 5 and 24 h. Dead cells were identified by Annexin V and PI staining. B) The expression level of MLKL protein in primary immune cells. MLKL expression was measured by Western blot analysis. C) Representative histograms were the MLKL expression of the indicated thymic and splenic subsets. Expression levels of MLKL were measured by flow cytometry. Total thymocytes and splenocytes were treated with TNF‐ α (50 ng mL −1 ), z‐VAD (20 µ m ) and Smac mimetic (200 n m ) for the indicated time or without, and cells were analyzed by Annexin V and Propidium Iodide (PI) staining and measured by D) Flow Cytometry. RIPK3, MLKL and p ‐MLKL protein was measured by E) Western blot analysis. Statistical analyses were performed using the two‐tailed unpaired Student t ‐test. P values below 0.05 were considered significant in the following manner: * p < 0.05, ** p < 0.01, *** p < 0.001.

    Techniques Used: Staining, Expressing, Western Blot, Flow Cytometry, Two Tailed Test

    Carcinogen‐induced thymic tumorigenesis was accelerated in RIPK3‐deficient mice. A) Intraperitoneal injection of ENU ( N ‐ethyl‐ N ‐nitrosourea) at around postnatal day 13 ∼15 (three‐time; 13∼15 day) was performed to both Ripk3 +/+ and Ripk3 −/− mice. B) Kaplan–Meier survival curve (Long‐rank test) after 3 consecutive daily ENU injections into Ripk3 wildtype and Ripk3 knockout mice ( n = 10 for each group). Around 105 days after ENU injection, 50% of Ripk3 −/− mice succumbed to tumors (red dotted line). C) All of animals were examined at 100 days after 3 consecutive daily ENU injection to Ripk3 +/+ ( n = 6) and Ripk3 −/− ( n = 6) mice. The summary of tumor incidence in this ENU injection experiment. 83% of the Ripk3 −/‐ animals developed thymic lymphoma at the microscopic level. D) Schematic diagram of an ENU injection experiment. E) Gross view of the abnormally big thymus removed from ENU injected Ripk3 +/+ and Ripk3 −/− mice at day 60. Ripk3 −/− thymus showed a more hyperplasia than Ripk3 +/+ thymus. F) Representative images of hematoxylin and eosin (H&E) stained sections and Ki‐67 staining of thymus from both ENU injected Ripk3 +/+ and Ripk3 −/− mice. G) Relative fluorescence of Ki‐67 in indicated thymic T cells (blue box in Ripk3 +/+ and red box in Ripk3 −/− ) analyzed by Flow Cytometry ( n = 10 ∼ 12 for each group). Statistical analyses were performed using the two‐tailed unpaired Student t ‐test or log rank test. P values below 0.05 were considered significant in the following manner: * p < 0.05, ** p < 0.01, *** p < 0.001.
    Figure Legend Snippet: Carcinogen‐induced thymic tumorigenesis was accelerated in RIPK3‐deficient mice. A) Intraperitoneal injection of ENU ( N ‐ethyl‐ N ‐nitrosourea) at around postnatal day 13 ∼15 (three‐time; 13∼15 day) was performed to both Ripk3 +/+ and Ripk3 −/− mice. B) Kaplan–Meier survival curve (Long‐rank test) after 3 consecutive daily ENU injections into Ripk3 wildtype and Ripk3 knockout mice ( n = 10 for each group). Around 105 days after ENU injection, 50% of Ripk3 −/− mice succumbed to tumors (red dotted line). C) All of animals were examined at 100 days after 3 consecutive daily ENU injection to Ripk3 +/+ ( n = 6) and Ripk3 −/− ( n = 6) mice. The summary of tumor incidence in this ENU injection experiment. 83% of the Ripk3 −/‐ animals developed thymic lymphoma at the microscopic level. D) Schematic diagram of an ENU injection experiment. E) Gross view of the abnormally big thymus removed from ENU injected Ripk3 +/+ and Ripk3 −/− mice at day 60. Ripk3 −/− thymus showed a more hyperplasia than Ripk3 +/+ thymus. F) Representative images of hematoxylin and eosin (H&E) stained sections and Ki‐67 staining of thymus from both ENU injected Ripk3 +/+ and Ripk3 −/− mice. G) Relative fluorescence of Ki‐67 in indicated thymic T cells (blue box in Ripk3 +/+ and red box in Ripk3 −/− ) analyzed by Flow Cytometry ( n = 10 ∼ 12 for each group). Statistical analyses were performed using the two‐tailed unpaired Student t ‐test or log rank test. P values below 0.05 were considered significant in the following manner: * p < 0.05, ** p < 0.01, *** p < 0.001.

    Techniques Used: Injection, Knock-Out, Staining, Fluorescence, Flow Cytometry, Two Tailed Test

    RIPK3 deficiency enhances thymic lymphoma in p53‐deficient mice via ERK hyperactivation. A) Kaplan‐Meier survival curve (Long‐rank test) of animals in different genetic backgrounds including Ripk3 +/+ p53 −/− and Ripk3 −/− p53 −/− animals. Red dotted line shows median survival of Ripk3 +/+ p53 −/− and Ripk3 −/− p53 −/‐ mice, which are significantly different ( p < 0.0001). B) The thymic lymphoma free survival curve and thymic lymphoma incidence of p53 −/− and Ripk3 −/− p53 −/− animals until 150 days of age. The tumor‐free survival plot of Ripk3 +/+ p53 −/− ( n = 42) compared with Ripk3 −/− p53 −/‐ ( n = 158) mice that are significantly different. C) Representative images of thymus, spleen and lymph node from tumor‐bearing Ripk3 +/+ p53 −/− and Ripk3 −/− p53 −/− littermates. D) The phenotype of the thymic T cell populations (left panel). The thymic T cell from both Ripk3 +/+ p53 −/− and Ripk3 −/− p53 −/− thymic lymphoma showed homogenous expansion in DP T cells but Ripk3 −/− p53 −/− DP T cells showed increased proliferation index (Ki‐67) (right panel). E) RNA sequencing analysis of up‐ and down‐regulated gene in Ripk3 −/− p53 −/− thymic lymphoma compared to p53 −/− thymic lymphoma. Analysis of up‐regulated gene shown the change in positive regulation of ERK signaling pathways. F) Total thymocytes from Ripk3 +/+ ( n = 4) and Ripk3 −/− ( n = 4) mice were stimulated with anti‐CD3 (1 µg mL −1 ) and anti‐CD28 (2 µg mL −1 ) or without for indicated time. Phosphorylation of ERK in DP T cell measured by Flow Cytometry. G) The protein expression levels of p‐ERK in normal thymus ( Ripk3 +/− p53 +/− ; n = 3 , Ripk3 −/− p53 +/+ ; n = 3) and thymic lymphoma ( Ripk3 +/+ p53 −/− ; n = 10 , Ripk3 −/− p53 −/− ; n = 10) tissues were measured by Western blot analysis. Statistical analyses were performed using the two‐tailed unpaired Student t ‐test or log rank test. p values below 0.05 were considered significant in the following manner: * p < 0.05, ** p < 0.01, *** p < 0.001.
    Figure Legend Snippet: RIPK3 deficiency enhances thymic lymphoma in p53‐deficient mice via ERK hyperactivation. A) Kaplan‐Meier survival curve (Long‐rank test) of animals in different genetic backgrounds including Ripk3 +/+ p53 −/− and Ripk3 −/− p53 −/− animals. Red dotted line shows median survival of Ripk3 +/+ p53 −/− and Ripk3 −/− p53 −/‐ mice, which are significantly different ( p < 0.0001). B) The thymic lymphoma free survival curve and thymic lymphoma incidence of p53 −/− and Ripk3 −/− p53 −/− animals until 150 days of age. The tumor‐free survival plot of Ripk3 +/+ p53 −/− ( n = 42) compared with Ripk3 −/− p53 −/‐ ( n = 158) mice that are significantly different. C) Representative images of thymus, spleen and lymph node from tumor‐bearing Ripk3 +/+ p53 −/− and Ripk3 −/− p53 −/− littermates. D) The phenotype of the thymic T cell populations (left panel). The thymic T cell from both Ripk3 +/+ p53 −/− and Ripk3 −/− p53 −/− thymic lymphoma showed homogenous expansion in DP T cells but Ripk3 −/− p53 −/− DP T cells showed increased proliferation index (Ki‐67) (right panel). E) RNA sequencing analysis of up‐ and down‐regulated gene in Ripk3 −/− p53 −/− thymic lymphoma compared to p53 −/− thymic lymphoma. Analysis of up‐regulated gene shown the change in positive regulation of ERK signaling pathways. F) Total thymocytes from Ripk3 +/+ ( n = 4) and Ripk3 −/− ( n = 4) mice were stimulated with anti‐CD3 (1 µg mL −1 ) and anti‐CD28 (2 µg mL −1 ) or without for indicated time. Phosphorylation of ERK in DP T cell measured by Flow Cytometry. G) The protein expression levels of p‐ERK in normal thymus ( Ripk3 +/− p53 +/− ; n = 3 , Ripk3 −/− p53 +/+ ; n = 3) and thymic lymphoma ( Ripk3 +/+ p53 −/− ; n = 10 , Ripk3 −/− p53 −/− ; n = 10) tissues were measured by Western blot analysis. Statistical analyses were performed using the two‐tailed unpaired Student t ‐test or log rank test. p values below 0.05 were considered significant in the following manner: * p < 0.05, ** p < 0.01, *** p < 0.001.

    Techniques Used: RNA Sequencing Assay, Flow Cytometry, Expressing, Western Blot, Two Tailed Test

    TCR‐mediated activation of LCK interacts with and RIPK3 to promote RIPK3 phosphorylation. A) Total thymocytes from Ripk3 +/+ ( n = 5) and Ripk3 −/− ( n = 5) mice were cultured with anti‐CD3 (1 µg mL −1 ) and anti‐CD28 (2 µg mL −1 ) or without for indicated time. Relative fluorescence of RIPK3 and p‐RIPK3 in DP T cells. B) Phosphorylation of RIPK3 increased in anti‐CD3 (1 µg mL −1 ) and anti‐CD28 (2 µg mL −1 ) stimulation. C) Experimental workflow for TAP (Tandem Affinity purification) pull‐down assay. An aliquot of each purified sample was loaded to SDS‐PAGE and stained with Coomassie brilliant blue. Potential RIPK3‐binding proteins were identified by LC‐MS/MS. Venn diagram represents the overlap of proteins and unique proteins identified by LC/MS/MS among TAP‐purified samples as indicated. Total 503 proteins were identified as specific RIPK3 binding proteins. There were several kinases and phosphatases. D) Computational docking model for RIPK3 (olive) and LCK (blue) predicted using ClusPro (see Materials and Methods). E) Western blot analysis after immunoprecipitation of mouse RIPK3 and mouse LCK in HEK293T cells. HEK293T cells were transfected with LCK and/or Flag‐RIPK3 expression constructs. Cells were harvested at 24 h after transfection. The endogenous RIPK3 interacted with LCK in response to anti‐CD3/CD28 stimulation. Interaction of RIPK3 and LCK was observed by F) Western blot analysis, and confirmed with G) Duolink proximity ligation assay. H) Tyrosine phosphorylation of RIPK3 were detected in LCK and Flag‐RIPK3 expressing HEK293T cells. Statistical analyses were performed using the two‐tailed unpaired Student t ‐test. p values below 0.05 were considered significant in the following manner: * p < 0.05, ** p < 0.01, *** p < 0.001.
    Figure Legend Snippet: TCR‐mediated activation of LCK interacts with and RIPK3 to promote RIPK3 phosphorylation. A) Total thymocytes from Ripk3 +/+ ( n = 5) and Ripk3 −/− ( n = 5) mice were cultured with anti‐CD3 (1 µg mL −1 ) and anti‐CD28 (2 µg mL −1 ) or without for indicated time. Relative fluorescence of RIPK3 and p‐RIPK3 in DP T cells. B) Phosphorylation of RIPK3 increased in anti‐CD3 (1 µg mL −1 ) and anti‐CD28 (2 µg mL −1 ) stimulation. C) Experimental workflow for TAP (Tandem Affinity purification) pull‐down assay. An aliquot of each purified sample was loaded to SDS‐PAGE and stained with Coomassie brilliant blue. Potential RIPK3‐binding proteins were identified by LC‐MS/MS. Venn diagram represents the overlap of proteins and unique proteins identified by LC/MS/MS among TAP‐purified samples as indicated. Total 503 proteins were identified as specific RIPK3 binding proteins. There were several kinases and phosphatases. D) Computational docking model for RIPK3 (olive) and LCK (blue) predicted using ClusPro (see Materials and Methods). E) Western blot analysis after immunoprecipitation of mouse RIPK3 and mouse LCK in HEK293T cells. HEK293T cells were transfected with LCK and/or Flag‐RIPK3 expression constructs. Cells were harvested at 24 h after transfection. The endogenous RIPK3 interacted with LCK in response to anti‐CD3/CD28 stimulation. Interaction of RIPK3 and LCK was observed by F) Western blot analysis, and confirmed with G) Duolink proximity ligation assay. H) Tyrosine phosphorylation of RIPK3 were detected in LCK and Flag‐RIPK3 expressing HEK293T cells. Statistical analyses were performed using the two‐tailed unpaired Student t ‐test. p values below 0.05 were considered significant in the following manner: * p < 0.05, ** p < 0.01, *** p < 0.001.

    Techniques Used: Activation Assay, Cell Culture, Fluorescence, Affinity Purification, Pull Down Assay, Purification, SDS Page, Staining, Binding Assay, Liquid Chromatography with Mass Spectroscopy, Western Blot, Immunoprecipitation, Transfection, Expressing, Construct, Proximity Ligation Assay, Two Tailed Test

    RIPK3 suppresses ERK phosphorylation by activating PP2A function. A) Western blot analysis of ERK phosphorylation after transiently expression of human RIPK3 and human LCK in HEK293T cells. HEK293T cells were transfected with GST‐LCK and/or Flag‐RIPK3 expression constructs. B) Computational docking model for RIPK3 (olive) and PP2A predicted using ClusPro. The PP2A comprises three subunits (A subunit; blue, B55 α subunit; green, and C subunit: magenta). C) Western blot analysis after immunoprecipitation of human RIPK3 and human PP2A subunits in HEK293T cells. HEK293T cells were transfected with GFP‐PPP2CA or GFP‐PPP2R2A and/or Flag‐RIPK3 expression constructs. Cells were harvested at 24 h after transfection. D) The endogenous RIPK3 interacted with PPP2R2A in response to anti‐CD3/CD28 stimulation. E) In vitro kinase activity of RIPK3 toward PPP2R2A with 32 P‐labeled ATP. To avoid false positive artifacts in the in vitro kinase assay, we included conditions with or without ATP. GST‐RIPK3 (amino acid 2–328) and GST‐PPP2R2A recombinant proteins were purified from sf‐9 cells. F) Total thymocytes from Ripk3 +/+ and Ripk3 −/− mice were stimulated with anti‐CD3 (1 µg mL −1 ) and anti‐CD28 (2 µg mL −1 ) for indicated time. Cell lysates were immunoprecipitated with anti‐ERK antibody. Immunocomplexes and cell lysates were analyzed by immunoblotting.
    Figure Legend Snippet: RIPK3 suppresses ERK phosphorylation by activating PP2A function. A) Western blot analysis of ERK phosphorylation after transiently expression of human RIPK3 and human LCK in HEK293T cells. HEK293T cells were transfected with GST‐LCK and/or Flag‐RIPK3 expression constructs. B) Computational docking model for RIPK3 (olive) and PP2A predicted using ClusPro. The PP2A comprises three subunits (A subunit; blue, B55 α subunit; green, and C subunit: magenta). C) Western blot analysis after immunoprecipitation of human RIPK3 and human PP2A subunits in HEK293T cells. HEK293T cells were transfected with GFP‐PPP2CA or GFP‐PPP2R2A and/or Flag‐RIPK3 expression constructs. Cells were harvested at 24 h after transfection. D) The endogenous RIPK3 interacted with PPP2R2A in response to anti‐CD3/CD28 stimulation. E) In vitro kinase activity of RIPK3 toward PPP2R2A with 32 P‐labeled ATP. To avoid false positive artifacts in the in vitro kinase assay, we included conditions with or without ATP. GST‐RIPK3 (amino acid 2–328) and GST‐PPP2R2A recombinant proteins were purified from sf‐9 cells. F) Total thymocytes from Ripk3 +/+ and Ripk3 −/− mice were stimulated with anti‐CD3 (1 µg mL −1 ) and anti‐CD28 (2 µg mL −1 ) for indicated time. Cell lysates were immunoprecipitated with anti‐ERK antibody. Immunocomplexes and cell lysates were analyzed by immunoblotting.

    Techniques Used: Western Blot, Expressing, Transfection, Construct, Immunoprecipitation, In Vitro, Activity Assay, Labeling, Kinase Assay, Recombinant, Purification

    Pharmacological modulation of PP2A activity regulates DP thymocyte hyperproliferation‐associated tumorigenesis. A) Intraperitoneal injection of ENU at around postnatal day 13 ∼15 (three‐time) was performed to Ripk3 +/+ mice. After 30 days of injection, mice were injected with LB‐100 or PBS intraperitoneally at 2 mg kg −1 on alternate days for 30 days. ( n = 4 for each group). B) Phosphorylation of ERK increased in response to anti‐CD3 (1 µg mL −1 ) and anti‐CD28 (2 µg mL −1 ) stimulation, which was further increased by LB‐100 treatment. C) Activation of ERK increased in LB‐100 injected Ripk3 +/+ thymus. D) Relative fluorescence of Ki‐67 in thymic T cells analyzed by Flow Cytometry. Ki‐67 expression is enhanced in LB‐100 injected Ripk3 +/+ thymocytes. E) Schematic diagram of an ENU injection experiment. After 30 days of ENU injection, Ripk3 −/− mice were injected with SMAP or PBS intraperitoneally at 2 mg kg −1 on alternate days for 30 days. ( n = 4 – 6 for each group). F) Activation of ERK reduced in SMAP injected Ripk3 −/− thymus. G) Ki‐67 expression is decreased in SMAP injected Ripk3 −/− thymocytes. Relative fluorescence of Ki‐67 expression analyzed by Flow Cytometry. Statistical analyses were performed using the two‐tailed unpaired Student t ‐test. P values below 0.05 were considered significant in the following manner: * p < 0.05, ** p < 0.01, *** p < 0.001.
    Figure Legend Snippet: Pharmacological modulation of PP2A activity regulates DP thymocyte hyperproliferation‐associated tumorigenesis. A) Intraperitoneal injection of ENU at around postnatal day 13 ∼15 (three‐time) was performed to Ripk3 +/+ mice. After 30 days of injection, mice were injected with LB‐100 or PBS intraperitoneally at 2 mg kg −1 on alternate days for 30 days. ( n = 4 for each group). B) Phosphorylation of ERK increased in response to anti‐CD3 (1 µg mL −1 ) and anti‐CD28 (2 µg mL −1 ) stimulation, which was further increased by LB‐100 treatment. C) Activation of ERK increased in LB‐100 injected Ripk3 +/+ thymus. D) Relative fluorescence of Ki‐67 in thymic T cells analyzed by Flow Cytometry. Ki‐67 expression is enhanced in LB‐100 injected Ripk3 +/+ thymocytes. E) Schematic diagram of an ENU injection experiment. After 30 days of ENU injection, Ripk3 −/− mice were injected with SMAP or PBS intraperitoneally at 2 mg kg −1 on alternate days for 30 days. ( n = 4 – 6 for each group). F) Activation of ERK reduced in SMAP injected Ripk3 −/− thymus. G) Ki‐67 expression is decreased in SMAP injected Ripk3 −/− thymocytes. Relative fluorescence of Ki‐67 expression analyzed by Flow Cytometry. Statistical analyses were performed using the two‐tailed unpaired Student t ‐test. P values below 0.05 were considered significant in the following manner: * p < 0.05, ** p < 0.01, *** p < 0.001.

    Techniques Used: Activity Assay, Injection, Activation Assay, Fluorescence, Flow Cytometry, Expressing, Two Tailed Test

    phospho ripk3 thr231 ser232 for mouse  (Abcam)

     
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    Structured Review

    Abcam phospho ripk3 thr231 ser232 for mouse
    (A) Representative immunoblot images of <t>RIPK3</t> phosphorylation <t>(Thr231/Ser232),</t> RIPK3, MLKL phosphorylation (Ser345), MLKL, RIPK1 phosphorylation (Ser166) protein and (B) quantification of RIPK3 phosphorylation, RIPK3, MLKL phosphorylation, RIPK1 phosphorylation protein levels in lung tissues from mice treated with oligomycin either control or LPS (n=3 per each group). β-actin or MLKL were used as loading control. Data are representative of three independent experiments. Data are mean±SD. * p<0.05; ** p<0.01; *** p<0.001 by Student’s 2-tailed t-test or ANOVA.
    Phospho Ripk3 Thr231 Ser232 For Mouse, supplied by Abcam, 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/phospho ripk3 thr231 ser232 for mouse/product/Abcam
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    phospho ripk3 thr231 ser232 for mouse - by Bioz Stars, 2024-09
    86/100 stars

    Images

    1) Product Images from "Impairment of Mitochondrial ATP Synthesis Induces RIPK3-dependent Necroptosis in Lung Epithelial Cells During Lung Injury by Lung Inflammation"

    Article Title: Impairment of Mitochondrial ATP Synthesis Induces RIPK3-dependent Necroptosis in Lung Epithelial Cells During Lung Injury by Lung Inflammation

    Journal: Immune Network

    doi: 10.4110/in.2022.22.e18

    (A) Representative immunoblot images of RIPK3 phosphorylation (Thr231/Ser232), RIPK3, MLKL phosphorylation (Ser345), MLKL, RIPK1 phosphorylation (Ser166) protein and (B) quantification of RIPK3 phosphorylation, RIPK3, MLKL phosphorylation, RIPK1 phosphorylation protein levels in lung tissues from mice treated with oligomycin either control or LPS (n=3 per each group). β-actin or MLKL were used as loading control. Data are representative of three independent experiments. Data are mean±SD. * p<0.05; ** p<0.01; *** p<0.001 by Student’s 2-tailed t-test or ANOVA.
    Figure Legend Snippet: (A) Representative immunoblot images of RIPK3 phosphorylation (Thr231/Ser232), RIPK3, MLKL phosphorylation (Ser345), MLKL, RIPK1 phosphorylation (Ser166) protein and (B) quantification of RIPK3 phosphorylation, RIPK3, MLKL phosphorylation, RIPK1 phosphorylation protein levels in lung tissues from mice treated with oligomycin either control or LPS (n=3 per each group). β-actin or MLKL were used as loading control. Data are representative of three independent experiments. Data are mean±SD. * p<0.05; ** p<0.01; *** p<0.001 by Student’s 2-tailed t-test or ANOVA.

    Techniques Used: Western Blot

    (A) Representative immunofluorescence images for MLKL phosphorylation at Ser345 (P-MLKL) staining (green) in lung epithelial cells expressing E-cadherin (red) of mice treated with oligomycin either control or LPS. Scale bars, 20 µm. DAPI-stained nuclei are shown in blue. White arrows indicate P-MLKL positive cells (n=10 images per individual subject, n=5 per each group). (B) Quantification of intensity for P-MLKL-positive staining and (C) quantification of P-MLKL and E-cadherin-positive cells from immunofluorescence images in A. (D) Representative immunoblot images of RIPK3 phosphorylation (Thr231/Ser232), RIPK3, MLKL phosphorylation (Ser345), MLKL, RIPK1 phosphorylation (Ser166) protein from lung tissue sections of A. MLKL was used as loading control. (E) Quantification of protein levels in BALF of lungs from mice treated with oligomycin either control or LPS (n=5 per each group). (F) Quantification of total inflammatory cells in BALF of lungs from mice treated with oligomycin either control or LPS (n=5 per each group). Data are representative of three independent experiments. Data are mean±SD. * p<0.05; ** p<0.01; *** p<0.001 by Student’s 2-tailed t-test or ANOVA.
    Figure Legend Snippet: (A) Representative immunofluorescence images for MLKL phosphorylation at Ser345 (P-MLKL) staining (green) in lung epithelial cells expressing E-cadherin (red) of mice treated with oligomycin either control or LPS. Scale bars, 20 µm. DAPI-stained nuclei are shown in blue. White arrows indicate P-MLKL positive cells (n=10 images per individual subject, n=5 per each group). (B) Quantification of intensity for P-MLKL-positive staining and (C) quantification of P-MLKL and E-cadherin-positive cells from immunofluorescence images in A. (D) Representative immunoblot images of RIPK3 phosphorylation (Thr231/Ser232), RIPK3, MLKL phosphorylation (Ser345), MLKL, RIPK1 phosphorylation (Ser166) protein from lung tissue sections of A. MLKL was used as loading control. (E) Quantification of protein levels in BALF of lungs from mice treated with oligomycin either control or LPS (n=5 per each group). (F) Quantification of total inflammatory cells in BALF of lungs from mice treated with oligomycin either control or LPS (n=5 per each group). Data are representative of three independent experiments. Data are mean±SD. * p<0.05; ** p<0.01; *** p<0.001 by Student’s 2-tailed t-test or ANOVA.

    Techniques Used: Immunofluorescence, Staining, Expressing, Western Blot

    (A) Representative immunohistochemistry images for RIPK3 staining in lung tissues from patients with ARDS (ARDS) and non-ARDS subjects (Normal). Scale bars, 100 µm. Black arrows indicate RIPK3-positive cells. (B) Quantification of intensity for RIPK3-positive staining in cells and (C) quantification of RIPK3-positive cells from immunohistochemistry images in A (ARDS, n=7; Normal, n=2; n = 10 images per individual subject). (D) Representative immunoblot images of RIPK3 phosphorylation (Ser227), RIPK3, MLKL phosphorylation (Ser358), MLKL, RIPK1 phosphorylation (Ser166) protein (left) and quantification of RIPK3 phosphorylation, RIPK3, MLKL phosphorylation, RIPK1 phosphorylation protein levels (right) in lung tissues from ARDS and Normal (n=2 per each group). β-actin or MLKL were used as loading control. Data are representative of three independent experiments. Data are mean±SD. ** p<0.01; *** p<0.001 by Student’s 2-tailed t-test or Mann–Whitney test.
    Figure Legend Snippet: (A) Representative immunohistochemistry images for RIPK3 staining in lung tissues from patients with ARDS (ARDS) and non-ARDS subjects (Normal). Scale bars, 100 µm. Black arrows indicate RIPK3-positive cells. (B) Quantification of intensity for RIPK3-positive staining in cells and (C) quantification of RIPK3-positive cells from immunohistochemistry images in A (ARDS, n=7; Normal, n=2; n = 10 images per individual subject). (D) Representative immunoblot images of RIPK3 phosphorylation (Ser227), RIPK3, MLKL phosphorylation (Ser358), MLKL, RIPK1 phosphorylation (Ser166) protein (left) and quantification of RIPK3 phosphorylation, RIPK3, MLKL phosphorylation, RIPK1 phosphorylation protein levels (right) in lung tissues from ARDS and Normal (n=2 per each group). β-actin or MLKL were used as loading control. Data are representative of three independent experiments. Data are mean±SD. ** p<0.01; *** p<0.001 by Student’s 2-tailed t-test or Mann–Whitney test.

    Techniques Used: Immunohistochemistry, Staining, Western Blot, MANN-WHITNEY

    (A) Representative immunofluorescence images for MLKL phosphorylation at Ser358 (P-MLKL) staining (green) in lung epithelial cells expressing E-cadherin (red) of lung tissues from patients with ARDS (ARDS) and non-ARDS subjects (Normal). Scale bars, 20 µm. DAPI-stained nuclei are shown in blue. (B) Quantification of intensity for P-MLKL-positive staining and (C) quantification of P-MLKL and E-cadherin-positive cells from immunofluorescence images in A (ARDS, n=7; Normal, n=2; n=10 images per individual subject). (D) Representative immunoblot images of RIPK3 phosphorylation (Ser227), RIPK3, MLKL phosphorylation (Ser358), MLKL, RIPK1 phosphorylation (Ser166) protein from lung tissue sections of A. MLKL was used as loading control. (E) A schematic diagram to summarize our new findings. Red arrow means an increase and blue arrow means a decrease in the diagram. Data are representative of 3 independent experiments. Data are mean ± SD. ** p<0.01 by Student’s 2-tailed t-test.
    Figure Legend Snippet: (A) Representative immunofluorescence images for MLKL phosphorylation at Ser358 (P-MLKL) staining (green) in lung epithelial cells expressing E-cadherin (red) of lung tissues from patients with ARDS (ARDS) and non-ARDS subjects (Normal). Scale bars, 20 µm. DAPI-stained nuclei are shown in blue. (B) Quantification of intensity for P-MLKL-positive staining and (C) quantification of P-MLKL and E-cadherin-positive cells from immunofluorescence images in A (ARDS, n=7; Normal, n=2; n=10 images per individual subject). (D) Representative immunoblot images of RIPK3 phosphorylation (Ser227), RIPK3, MLKL phosphorylation (Ser358), MLKL, RIPK1 phosphorylation (Ser166) protein from lung tissue sections of A. MLKL was used as loading control. (E) A schematic diagram to summarize our new findings. Red arrow means an increase and blue arrow means a decrease in the diagram. Data are representative of 3 independent experiments. Data are mean ± SD. ** p<0.01 by Student’s 2-tailed t-test.

    Techniques Used: Immunofluorescence, Staining, Expressing, Western Blot

    mouse p ripk3  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc mouse p ripk3
    Mouse P Ripk3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rabbit anti mouse phospho ripk3 thr231 ser232 monoclonal antibody  (Abcam)

     
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    Abcam rabbit anti mouse phospho ripk3 thr231 ser232 monoclonal antibody
    Immunohistochemical examination for proliferation rate (labelled by PCNA-positive cells), <t>RIPK3-</t> and Phospho-RIPK3 positive cells in the gastric fundus of FVB/N mice at an age of 6 months and gastric body of human stomach. In the stomach of FVB/N mice at the age of 6 months, image ( A ) showed a higher rate of RIPK3-positive cells on fundus glands (arrow in A ) and some on surface mucous cells (arrowhead in A ). B showed that RIPK3-positive cells were mostly located in in the fundus glands and distributed in the isthmus and neck regions (arrow in B ), and few in the base regions. C showed positive cells for active form of RIPK3 (phosphor-RIPK3) in a low density in the mouse gastric glandular cells. In the body of human stomach, image ( D ) visualized that PCNA-positive cells were observed in both surface mucous cells (arrowhead in D ) and gastric glands (arrow in D ). RIPK3-positive cells were also shown in the same compartments of gastric body ( E ). However, RIPK3-positive cells were evenly distributed in the glands. F was the negative isotopic-matched control. ( A – F , IHC images. Counterstained with Hematoxylin, original magnification 400 ×)
    Rabbit Anti Mouse Phospho Ripk3 Thr231 Ser232 Monoclonal Antibody, supplied by Abcam, 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 expression of RIPK3 is associated with cell turnover of gastric mucosa in the mouse and human stomach"

    Article Title: The expression of RIPK3 is associated with cell turnover of gastric mucosa in the mouse and human stomach

    Journal: Journal of Molecular Histology

    doi: 10.1007/s10735-021-10001-5

    Immunohistochemical examination for proliferation rate (labelled by PCNA-positive cells), RIPK3- and Phospho-RIPK3 positive cells in the gastric fundus of FVB/N mice at an age of 6 months and gastric body of human stomach. In the stomach of FVB/N mice at the age of 6 months, image ( A ) showed a higher rate of RIPK3-positive cells on fundus glands (arrow in A ) and some on surface mucous cells (arrowhead in A ). B showed that RIPK3-positive cells were mostly located in in the fundus glands and distributed in the isthmus and neck regions (arrow in B ), and few in the base regions. C showed positive cells for active form of RIPK3 (phosphor-RIPK3) in a low density in the mouse gastric glandular cells. In the body of human stomach, image ( D ) visualized that PCNA-positive cells were observed in both surface mucous cells (arrowhead in D ) and gastric glands (arrow in D ). RIPK3-positive cells were also shown in the same compartments of gastric body ( E ). However, RIPK3-positive cells were evenly distributed in the glands. F was the negative isotopic-matched control. ( A – F , IHC images. Counterstained with Hematoxylin, original magnification 400 ×)
    Figure Legend Snippet: Immunohistochemical examination for proliferation rate (labelled by PCNA-positive cells), RIPK3- and Phospho-RIPK3 positive cells in the gastric fundus of FVB/N mice at an age of 6 months and gastric body of human stomach. In the stomach of FVB/N mice at the age of 6 months, image ( A ) showed a higher rate of RIPK3-positive cells on fundus glands (arrow in A ) and some on surface mucous cells (arrowhead in A ). B showed that RIPK3-positive cells were mostly located in in the fundus glands and distributed in the isthmus and neck regions (arrow in B ), and few in the base regions. C showed positive cells for active form of RIPK3 (phosphor-RIPK3) in a low density in the mouse gastric glandular cells. In the body of human stomach, image ( D ) visualized that PCNA-positive cells were observed in both surface mucous cells (arrowhead in D ) and gastric glands (arrow in D ). RIPK3-positive cells were also shown in the same compartments of gastric body ( E ). However, RIPK3-positive cells were evenly distributed in the glands. F was the negative isotopic-matched control. ( A – F , IHC images. Counterstained with Hematoxylin, original magnification 400 ×)

    Techniques Used: Immunohistochemical staining

    Counting data of PCNA- and RIPK3-positive cells in the fundus glands of 6-month-old FVB/N mice and body glands of human stomachs. It showed rates of PCNA-positive cell/gland ( White bar) and RIPK3-positive cells/gland ( Grey bar) in the fundus of mice ( A ) and the body of human stomach ( B ) respectively. The number of PCNA-positive cells/gland was higher than that of RIPK3-positive cells/gland and the PCNA/RIPK3 ratio was ~ 1.6 in the fundic glands of mice ( Black bar in A ), and the ratio was ~ 4.7 in the body glands of human ( Black bar in B )
    Figure Legend Snippet: Counting data of PCNA- and RIPK3-positive cells in the fundus glands of 6-month-old FVB/N mice and body glands of human stomachs. It showed rates of PCNA-positive cell/gland ( White bar) and RIPK3-positive cells/gland ( Grey bar) in the fundus of mice ( A ) and the body of human stomach ( B ) respectively. The number of PCNA-positive cells/gland was higher than that of RIPK3-positive cells/gland and the PCNA/RIPK3 ratio was ~ 1.6 in the fundic glands of mice ( Black bar in A ), and the ratio was ~ 4.7 in the body glands of human ( Black bar in B )

    Techniques Used:

    Double immunofluorescence examination on the expression of RIPK3 in glandular parietal cells in FVB/N mice and human. In the mouse gastric fundic section, RIPK3 (labelled by FITC, green color in B ) was shown in H + K + -ATPase positive glandular parietal cells (labelled by Alexa-647, red color in A , C for merged RIPK3/H + K + -ATPase). In the human gastric body section, similar colocalization of RIPK3 (labeled by FITC, green color in E ) with H + K + -ATPase-positive glandular parietal cells (labelled by Alexa-647, red color in D ) was observed ( F for merged RIPK3/H + K + -ATPase images). ( A – F , double immunofluorescence staining, original magnification 200×). (Color figure online)
    Figure Legend Snippet: Double immunofluorescence examination on the expression of RIPK3 in glandular parietal cells in FVB/N mice and human. In the mouse gastric fundic section, RIPK3 (labelled by FITC, green color in B ) was shown in H + K + -ATPase positive glandular parietal cells (labelled by Alexa-647, red color in A , C for merged RIPK3/H + K + -ATPase). In the human gastric body section, similar colocalization of RIPK3 (labeled by FITC, green color in E ) with H + K + -ATPase-positive glandular parietal cells (labelled by Alexa-647, red color in D ) was observed ( F for merged RIPK3/H + K + -ATPase images). ( A – F , double immunofluorescence staining, original magnification 200×). (Color figure online)

    Techniques Used: Immunofluorescence, Expressing, Labeling, Double Immunofluorescence Staining

    Double immunofluorescence examination of RIPK3-positive cell phenotypes in human gastric lamina propria. Phenotypic analysis revealed that RIPK3-positive cells ( B , E & H ) located in the lamina propria of human stomach were CD3-positive ( A ) lymphocytes (merged image, C ), CD34-positive ( D ) stromal cells (merged image, F ) and SMA-α-positive ( G ) stromal cells (merged image, I ). ( A – L , double immunofluorescence-stained confocal images, original magnification 200×)
    Figure Legend Snippet: Double immunofluorescence examination of RIPK3-positive cell phenotypes in human gastric lamina propria. Phenotypic analysis revealed that RIPK3-positive cells ( B , E & H ) located in the lamina propria of human stomach were CD3-positive ( A ) lymphocytes (merged image, C ), CD34-positive ( D ) stromal cells (merged image, F ) and SMA-α-positive ( G ) stromal cells (merged image, I ). ( A – L , double immunofluorescence-stained confocal images, original magnification 200×)

    Techniques Used: Immunofluorescence, Staining

    mouse p ripk3  (Cell Signaling Technology Inc)


    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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    Cell Signaling Technology Inc mouse p ripk3
    a , b Complex IIa was immunoprecipitated by FADD antibodies and analyzed by western blotting in Ripk1 +/+ and Ripk1 K612R/K612R MEFs pretreated with SM-164 (100 nM) ( a ) or 5Z-7 (200 nM) ( b ) for 2 h, and then treated with mTNFα (100 ng/ml) for different time points in the presence or absence of Nec-1s (10 μM) as indicated. Uncropped blots in the Source Data file. c , d Complex IIb was immunoprecipitated by <t>RIPK3</t> antibodies and analyzed by western blotting in Ripk1 +/+ and Ripk1 K612R/K612R MEFs pretreated with SM-164 (100 nM) ( c ), 5Z-7 (200 nM) ( d ) for 2 h, and then treated with mTNFα (100 ng/ml)/Z-VAD (25 μM) for indicated time. Uncropped blots in the Source Data file. e Complex I in Ripk1 +/+ and Ripk1 K612R/K612R MEFs treated with mTNFα (100 ng/ml) for indicated time points was immunoprecipitated with TNFR1 antibody and analyzed by western blotting. Uncropped blots in the Source Data file. f , g Complex I in Ripk1 +/+ and Ripk1 K612R/K612R MEFs pretreated with SM-164 (100 nM) ( f ) or 5Z-7 (200 nM) ( g ) for 2 h, and then treated with flag-mTNFα (100 ng/ml) for indicated time points was immunoprecipitated with Flag M2 Agarose Affinity gel and analyzed by western blotting in. Uncropped blots in the Source Data file.
    Mouse P Ripk3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Ubiquitination of RIPK1 regulates its activation mediated by TNFR1 and TLRs signaling in distinct manners"

    Article Title: Ubiquitination of RIPK1 regulates its activation mediated by TNFR1 and TLRs signaling in distinct manners

    Journal: Nature Communications

    doi: 10.1038/s41467-020-19935-y

    a , b Complex IIa was immunoprecipitated by FADD antibodies and analyzed by western blotting in Ripk1 +/+ and Ripk1 K612R/K612R MEFs pretreated with SM-164 (100 nM) ( a ) or 5Z-7 (200 nM) ( b ) for 2 h, and then treated with mTNFα (100 ng/ml) for different time points in the presence or absence of Nec-1s (10 μM) as indicated. Uncropped blots in the Source Data file. c , d Complex IIb was immunoprecipitated by RIPK3 antibodies and analyzed by western blotting in Ripk1 +/+ and Ripk1 K612R/K612R MEFs pretreated with SM-164 (100 nM) ( c ), 5Z-7 (200 nM) ( d ) for 2 h, and then treated with mTNFα (100 ng/ml)/Z-VAD (25 μM) for indicated time. Uncropped blots in the Source Data file. e Complex I in Ripk1 +/+ and Ripk1 K612R/K612R MEFs treated with mTNFα (100 ng/ml) for indicated time points was immunoprecipitated with TNFR1 antibody and analyzed by western blotting. Uncropped blots in the Source Data file. f , g Complex I in Ripk1 +/+ and Ripk1 K612R/K612R MEFs pretreated with SM-164 (100 nM) ( f ) or 5Z-7 (200 nM) ( g ) for 2 h, and then treated with flag-mTNFα (100 ng/ml) for indicated time points was immunoprecipitated with Flag M2 Agarose Affinity gel and analyzed by western blotting in. Uncropped blots in the Source Data file.
    Figure Legend Snippet: a , b Complex IIa was immunoprecipitated by FADD antibodies and analyzed by western blotting in Ripk1 +/+ and Ripk1 K612R/K612R MEFs pretreated with SM-164 (100 nM) ( a ) or 5Z-7 (200 nM) ( b ) for 2 h, and then treated with mTNFα (100 ng/ml) for different time points in the presence or absence of Nec-1s (10 μM) as indicated. Uncropped blots in the Source Data file. c , d Complex IIb was immunoprecipitated by RIPK3 antibodies and analyzed by western blotting in Ripk1 +/+ and Ripk1 K612R/K612R MEFs pretreated with SM-164 (100 nM) ( c ), 5Z-7 (200 nM) ( d ) for 2 h, and then treated with mTNFα (100 ng/ml)/Z-VAD (25 μM) for indicated time. Uncropped blots in the Source Data file. e Complex I in Ripk1 +/+ and Ripk1 K612R/K612R MEFs treated with mTNFα (100 ng/ml) for indicated time points was immunoprecipitated with TNFR1 antibody and analyzed by western blotting. Uncropped blots in the Source Data file. f , g Complex I in Ripk1 +/+ and Ripk1 K612R/K612R MEFs pretreated with SM-164 (100 nM) ( f ) or 5Z-7 (200 nM) ( g ) for 2 h, and then treated with flag-mTNFα (100 ng/ml) for indicated time points was immunoprecipitated with Flag M2 Agarose Affinity gel and analyzed by western blotting in. Uncropped blots in the Source Data file.

    Techniques Used: Immunoprecipitation, Western Blot

    a Primary Ripk1 +/+ and Ripk1 K612R/K612R BMDMs were pretreated with Nec-1s (10 μM), GSK’872 (10 μM) or vehicle in the presence or absence of Z-VAD (25 μM) for 30 min respectively, and then treated with Poly (I:C) (20 μg/ml) or LPS (50 ng/ml) as indicated for 10 h. Cell survival was measured by CellTiterGlo. Data are presented as mean ± SEM of n = 3 biologically independent samples. Two-way ANOVA with Bonferroni’s multiple comparison test. b , c Complex IIb was isolated by RIPK3 antibody and analyzed by western blotting in primary BMDMs isolated from indicated mouse strains pretreated with Z-VAD (25 μM) or vehicle for 30 min, and then treated with LPS (50 ng/ml) for indicated time points. Uncropped blots in the Source Data file. d Quantification of IL-1β and TNFα in the cultural supernatant from primary Ripk1 +/+ and Ripk1 K612R/K612R BMDMs treated with LPS (50 ng/ml) for indicated time points by ELISA. e Western blotting analysis of the cell lysates and cultural supernatant of Ripk1 +/+ and Ripk1 K612R/K612R primary BMDMs treated with LPS (100 ng/ml) for indicated time points. Uncropped blots in the Source Data file. f Primary BMDMs isolated from indicated mouse strains were treated with LPS (50 ng/ml) plus Z-VAD (25 μM) or Poly (I:C) (20 μg/ml) or plus Z-VAD (25 μM) as indicated for 10 h. Cell survival was measured by Cell TiterGlo. Data are presented as Mean±SEM of n = 3 biologically independent samples. Two-way ANOVA with Bonferroni’s multiple comparison test. g Western blotting analysis of primary BMDMs isolated from indicated mouse strains were treated with LPS (50 ng/ml) plus Z-VAD (25 μM) for indicated time points. Uncropped blots in the Source Data file. h , i Quantification of IL-1β in cultural supernatant by ELISA ( h ) and western blotting analysis of cell lysates and cultural supernatant ( i ) of primary BMDMs with indicated genotypes treated with LPS(50 ng/ml) for indicated time points. Uncropped blots in the Source Data file.
    Figure Legend Snippet: a Primary Ripk1 +/+ and Ripk1 K612R/K612R BMDMs were pretreated with Nec-1s (10 μM), GSK’872 (10 μM) or vehicle in the presence or absence of Z-VAD (25 μM) for 30 min respectively, and then treated with Poly (I:C) (20 μg/ml) or LPS (50 ng/ml) as indicated for 10 h. Cell survival was measured by CellTiterGlo. Data are presented as mean ± SEM of n = 3 biologically independent samples. Two-way ANOVA with Bonferroni’s multiple comparison test. b , c Complex IIb was isolated by RIPK3 antibody and analyzed by western blotting in primary BMDMs isolated from indicated mouse strains pretreated with Z-VAD (25 μM) or vehicle for 30 min, and then treated with LPS (50 ng/ml) for indicated time points. Uncropped blots in the Source Data file. d Quantification of IL-1β and TNFα in the cultural supernatant from primary Ripk1 +/+ and Ripk1 K612R/K612R BMDMs treated with LPS (50 ng/ml) for indicated time points by ELISA. e Western blotting analysis of the cell lysates and cultural supernatant of Ripk1 +/+ and Ripk1 K612R/K612R primary BMDMs treated with LPS (100 ng/ml) for indicated time points. Uncropped blots in the Source Data file. f Primary BMDMs isolated from indicated mouse strains were treated with LPS (50 ng/ml) plus Z-VAD (25 μM) or Poly (I:C) (20 μg/ml) or plus Z-VAD (25 μM) as indicated for 10 h. Cell survival was measured by Cell TiterGlo. Data are presented as Mean±SEM of n = 3 biologically independent samples. Two-way ANOVA with Bonferroni’s multiple comparison test. g Western blotting analysis of primary BMDMs isolated from indicated mouse strains were treated with LPS (50 ng/ml) plus Z-VAD (25 μM) for indicated time points. Uncropped blots in the Source Data file. h , i Quantification of IL-1β in cultural supernatant by ELISA ( h ) and western blotting analysis of cell lysates and cultural supernatant ( i ) of primary BMDMs with indicated genotypes treated with LPS(50 ng/ml) for indicated time points. Uncropped blots in the Source Data file.

    Techniques Used: Isolation, Western Blot, Enzyme-linked Immunosorbent Assay

    a , b Immunoprecipitation and western blotting analysis of RIPK1 interaction in HEK293T cells cotransfected with expression vectors of myc-hRIPK1-WT-FL, myc-RIPK1-hK627R-FL, myc-hRIPK1-DD-WT, myc-hRIPK1-DD-K627R, and GFP-hRIPK1-DD-WT ( a ) or that of myc-hRIPK1-WT, myc-hRIPK1-K627R, myc-hRIPK1-ΔDD, myc-hRIPK1-WT-4A, myc-hRIPK1-K627R-4A, myc-hRIPK1-ΔDD-4A, and HA-hRIPK1-WT ( b ) as indicated for 18 h. The cells lysates were immunoprecipitated with anti-Myc conjugated agarose beads and analyzed by western blotting using indicated antibody. Uncropped blots in the Source Data file. c , d MEF Ripk1 − / − cells were reconstituted with 2xFV-mRIPK1-WT or 2xFV-mRIPK1-K612R by PMSCV retrovirus infection. The cells were pretreated with Nec-1s (10 μM), GSK’872 (10 μM) or vehicle for 30 min, and then treated with AP20187 (30 nM) in the presence of Z-VAD (25 μM) for indicated time. Cell survival was measured by Cell TiterGlo ( c ). Cell lysates were analyzed by western blotting using indicated antibodies ( d ). AP20187 (30 nM) was added to induce dimerization. Uncropped blots in the Source Data file. e Immunoprecipitation and western blotting analysis of DD interaction in HEK293T cells cotransfected expression plasmids of myc-hRIPK1-WT, myc-hRIPK1-K627R, myc-hRIPK1-ΔDD with flag-hTNFR1-DD, flag-hTRADD-DD, or flag-hFADD-DD as indicated. Cells lysates were immunoprecipitated with Flag M2 Agarose Affinity gel and analyzed by western blotting using indicated antibodies. Uncropped blots in the Source Data file. f , g RIPK1 +/+ and RIPK1 K612R MEFs were reconstituted with 2xFV-RIPK3 by PMSCV retrovirus infection. Cells were pretreated with Nec-1s (10 μM), GSK’872 (10 μM) or vehicle for 30 min, and then treated with AP20187 (30 nM) in the presence of Z-VAD (25 μM) for indicated time to induce cell death. Cell survival was measured by Cell TiterGlo ( f ). Data are presented as mean ± SEM of n = 3 biologically independent samples. Two-way ANOVA with Bonferroni’s multiple comparison test. The cells were lysed in SDS reducing sample buffer and analyzed by western blotting using indicated antibodies ( g ). Uncropped blots in the Source Data file. h , i FADD was deleted in MEF cells stably expressing 2xFV-RIPK3 by means of CRISPR/Cas9. The cells were treated with AP20187 (30 nM) in the presence of Z-VAD (25 μM) for indicated time. Cell survival was measured by CellTiterGlo ( h ). Data are presented as mean ± SEM of n = 3 biologically independent samples. Two-way ANOVA with Bonferroni’s multiple comparison test. Cells were lysed in SDS reducing sample buffer and analyzed by western blotting using indicated antibodies ( i ). Uncropped blots in the Source Data file.
    Figure Legend Snippet: a , b Immunoprecipitation and western blotting analysis of RIPK1 interaction in HEK293T cells cotransfected with expression vectors of myc-hRIPK1-WT-FL, myc-RIPK1-hK627R-FL, myc-hRIPK1-DD-WT, myc-hRIPK1-DD-K627R, and GFP-hRIPK1-DD-WT ( a ) or that of myc-hRIPK1-WT, myc-hRIPK1-K627R, myc-hRIPK1-ΔDD, myc-hRIPK1-WT-4A, myc-hRIPK1-K627R-4A, myc-hRIPK1-ΔDD-4A, and HA-hRIPK1-WT ( b ) as indicated for 18 h. The cells lysates were immunoprecipitated with anti-Myc conjugated agarose beads and analyzed by western blotting using indicated antibody. Uncropped blots in the Source Data file. c , d MEF Ripk1 − / − cells were reconstituted with 2xFV-mRIPK1-WT or 2xFV-mRIPK1-K612R by PMSCV retrovirus infection. The cells were pretreated with Nec-1s (10 μM), GSK’872 (10 μM) or vehicle for 30 min, and then treated with AP20187 (30 nM) in the presence of Z-VAD (25 μM) for indicated time. Cell survival was measured by Cell TiterGlo ( c ). Cell lysates were analyzed by western blotting using indicated antibodies ( d ). AP20187 (30 nM) was added to induce dimerization. Uncropped blots in the Source Data file. e Immunoprecipitation and western blotting analysis of DD interaction in HEK293T cells cotransfected expression plasmids of myc-hRIPK1-WT, myc-hRIPK1-K627R, myc-hRIPK1-ΔDD with flag-hTNFR1-DD, flag-hTRADD-DD, or flag-hFADD-DD as indicated. Cells lysates were immunoprecipitated with Flag M2 Agarose Affinity gel and analyzed by western blotting using indicated antibodies. Uncropped blots in the Source Data file. f , g RIPK1 +/+ and RIPK1 K612R MEFs were reconstituted with 2xFV-RIPK3 by PMSCV retrovirus infection. Cells were pretreated with Nec-1s (10 μM), GSK’872 (10 μM) or vehicle for 30 min, and then treated with AP20187 (30 nM) in the presence of Z-VAD (25 μM) for indicated time to induce cell death. Cell survival was measured by Cell TiterGlo ( f ). Data are presented as mean ± SEM of n = 3 biologically independent samples. Two-way ANOVA with Bonferroni’s multiple comparison test. The cells were lysed in SDS reducing sample buffer and analyzed by western blotting using indicated antibodies ( g ). Uncropped blots in the Source Data file. h , i FADD was deleted in MEF cells stably expressing 2xFV-RIPK3 by means of CRISPR/Cas9. The cells were treated with AP20187 (30 nM) in the presence of Z-VAD (25 μM) for indicated time. Cell survival was measured by CellTiterGlo ( h ). Data are presented as mean ± SEM of n = 3 biologically independent samples. Two-way ANOVA with Bonferroni’s multiple comparison test. Cells were lysed in SDS reducing sample buffer and analyzed by western blotting using indicated antibodies ( i ). Uncropped blots in the Source Data file.

    Techniques Used: Immunoprecipitation, Western Blot, Expressing, Infection, Stable Transfection, CRISPR

    In TNFα-stimulated WT cells, RIPK1 is rapidly recruited to TNFR1 by direct binding to the death domain (DD) of TNFR1 or that of TRADD to initiate the formation of complex I. In complex I the ubiquitination of RIPK1 is modulated by multiple E3 ubiquitin ligases such as cIAP1/2 and LUBAC complex. Ubiquitination of RIPK1 mediates NF-κB activation. Ubiquitination of RIPK1 K612 modulates overall patterns of RIPK1 ubiquitination and promotes RIPK1-RIPK1 and RIPK1-TNFR1 interaction mediated by its DD, which leads to its activation and interaction with FADD/Caspase-8 to mediate RDA or with RIPK3 to mediate necroptosis when caspase-8 is inactivated. K612R mutation reduces the recruitment of RIPK1 and inhibits the activation of RIPK1 upon stimulation of TNFR1 by TNFα. RIPK1 K612R mutation inhibits cell death induced by TNFα through disrupting RIPK1 dimerization and RIPK1-TNFR1 interaction mediated by DD domain. In LPS or Poly (I:C)-stimulated WT cells, RIPK1 and RIPK3 interact with TRIF which regulates the activation of RIPK1 and RIPK3. Ubiquitination of RIPK1 K612 promotes RIPK1 ubiquitination, dimerization and interaction of RIPK1 with FADD, which in turn recruits FADD to suppress necroptosis and caspase-1 activation by restricting RIPK3 activation in response to TLR3 and TLR4. RIPK1 K612R mutation disrupts the interaction of RIPK1 and FADD to promote RIPK3-dependent necroptosis and inflammation induced by LPS or Poly(I:C).
    Figure Legend Snippet: In TNFα-stimulated WT cells, RIPK1 is rapidly recruited to TNFR1 by direct binding to the death domain (DD) of TNFR1 or that of TRADD to initiate the formation of complex I. In complex I the ubiquitination of RIPK1 is modulated by multiple E3 ubiquitin ligases such as cIAP1/2 and LUBAC complex. Ubiquitination of RIPK1 mediates NF-κB activation. Ubiquitination of RIPK1 K612 modulates overall patterns of RIPK1 ubiquitination and promotes RIPK1-RIPK1 and RIPK1-TNFR1 interaction mediated by its DD, which leads to its activation and interaction with FADD/Caspase-8 to mediate RDA or with RIPK3 to mediate necroptosis when caspase-8 is inactivated. K612R mutation reduces the recruitment of RIPK1 and inhibits the activation of RIPK1 upon stimulation of TNFR1 by TNFα. RIPK1 K612R mutation inhibits cell death induced by TNFα through disrupting RIPK1 dimerization and RIPK1-TNFR1 interaction mediated by DD domain. In LPS or Poly (I:C)-stimulated WT cells, RIPK1 and RIPK3 interact with TRIF which regulates the activation of RIPK1 and RIPK3. Ubiquitination of RIPK1 K612 promotes RIPK1 ubiquitination, dimerization and interaction of RIPK1 with FADD, which in turn recruits FADD to suppress necroptosis and caspase-1 activation by restricting RIPK3 activation in response to TLR3 and TLR4. RIPK1 K612R mutation disrupts the interaction of RIPK1 and FADD to promote RIPK3-dependent necroptosis and inflammation induced by LPS or Poly(I:C).

    Techniques Used: Binding Assay, Activation Assay, Mutagenesis

    rabbit anti phospho ripk3 thr231 ser232  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit anti phospho ripk3 thr231 ser232
    Rabbit Anti Phospho Ripk3 Thr231 Ser232, supplied by Cell Signaling Technology Inc, 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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    Genetic loss of <t>RIPK3</t> leads to hyperproliferation of DP thymocytes contributing to thymic lymphoma. A) Kaplan–Meier survival curve (Long‐rank test) in Ripk3 +/+ ( n = 51) and Ripk3 −/− ( n = 40) mice. B) Spontaneous tumors found in various organs and their frequency in Ripk3 +/+ and Ripk3 −/− animals. C) Thymus from Ripk3 +/+ and Ripk3 −/− was shown (left panel) and its weight was shown in the graph (right panel). ( n = 13 for each group). D) Representative H&E images of thymus from both Ripk3 +/+ and Ripk3 −/− mice. E) Percentage of the thymic T cell subsets (left panel) and the absolute cell number (right panel) by the indicated thymic subsets are shown in each graph. ( n = 7 for each group). F) Relative fluorescence of Ki‐67 in CD4 + CD8 + double positive (DP) T cells under anti‐CD3/CD28 stimulation. Total thymocytes from Ripk3 +/+ ( n = 5) and Ripk3 −/− ( n = 5) mice were stimulated with anti‐CD3 (1 µg mL −1 ) and anti‐CD28 (2 µg mL −1 ) or without, and then Ki‐67 in DP cell was measured by FACS. Statistical analyses were performed using the two‐tailed unpaired Student t ‐test. P values below 0.05 were considered significant in the following manner: * p < 0.05, ** p < 0.01, *** p < 0.001.
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    phospho ripk3 thr231 ser232 for mouse  (Abcam)
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    (A) Representative immunoblot images of <t>RIPK3</t> phosphorylation <t>(Thr231/Ser232),</t> RIPK3, MLKL phosphorylation (Ser345), MLKL, RIPK1 phosphorylation (Ser166) protein and (B) quantification of RIPK3 phosphorylation, RIPK3, MLKL phosphorylation, RIPK1 phosphorylation protein levels in lung tissues from mice treated with oligomycin either control or LPS (n=3 per each group). β-actin or MLKL were used as loading control. Data are representative of three independent experiments. Data are mean±SD. * p<0.05; ** p<0.01; *** p<0.001 by Student’s 2-tailed t-test or ANOVA.
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    rabbit anti mouse phospho ripk3 thr231 ser232 monoclonal antibody  (Abcam)
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    Immunohistochemical examination for proliferation rate (labelled by PCNA-positive cells), <t>RIPK3-</t> and Phospho-RIPK3 positive cells in the gastric fundus of FVB/N mice at an age of 6 months and gastric body of human stomach. In the stomach of FVB/N mice at the age of 6 months, image ( A ) showed a higher rate of RIPK3-positive cells on fundus glands (arrow in A ) and some on surface mucous cells (arrowhead in A ). B showed that RIPK3-positive cells were mostly located in in the fundus glands and distributed in the isthmus and neck regions (arrow in B ), and few in the base regions. C showed positive cells for active form of RIPK3 (phosphor-RIPK3) in a low density in the mouse gastric glandular cells. In the body of human stomach, image ( D ) visualized that PCNA-positive cells were observed in both surface mucous cells (arrowhead in D ) and gastric glands (arrow in D ). RIPK3-positive cells were also shown in the same compartments of gastric body ( E ). However, RIPK3-positive cells were evenly distributed in the glands. F was the negative isotopic-matched control. ( A – F , IHC images. Counterstained with Hematoxylin, original magnification 400 ×)
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    rabbit anti phospho ripk3 thr231 ser232  (Cell Signaling Technology Inc)
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    Immunohistochemical examination for proliferation rate (labelled by PCNA-positive cells), <t>RIPK3-</t> and Phospho-RIPK3 positive cells in the gastric fundus of FVB/N mice at an age of 6 months and gastric body of human stomach. In the stomach of FVB/N mice at the age of 6 months, image ( A ) showed a higher rate of RIPK3-positive cells on fundus glands (arrow in A ) and some on surface mucous cells (arrowhead in A ). B showed that RIPK3-positive cells were mostly located in in the fundus glands and distributed in the isthmus and neck regions (arrow in B ), and few in the base regions. C showed positive cells for active form of RIPK3 (phosphor-RIPK3) in a low density in the mouse gastric glandular cells. In the body of human stomach, image ( D ) visualized that PCNA-positive cells were observed in both surface mucous cells (arrowhead in D ) and gastric glands (arrow in D ). RIPK3-positive cells were also shown in the same compartments of gastric body ( E ). However, RIPK3-positive cells were evenly distributed in the glands. F was the negative isotopic-matched control. ( A – F , IHC images. Counterstained with Hematoxylin, original magnification 400 ×)
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    Genetic loss of RIPK3 leads to hyperproliferation of DP thymocytes contributing to thymic lymphoma. A) Kaplan–Meier survival curve (Long‐rank test) in Ripk3 +/+ ( n = 51) and Ripk3 −/− ( n = 40) mice. B) Spontaneous tumors found in various organs and their frequency in Ripk3 +/+ and Ripk3 −/− animals. C) Thymus from Ripk3 +/+ and Ripk3 −/− was shown (left panel) and its weight was shown in the graph (right panel). ( n = 13 for each group). D) Representative H&E images of thymus from both Ripk3 +/+ and Ripk3 −/− mice. E) Percentage of the thymic T cell subsets (left panel) and the absolute cell number (right panel) by the indicated thymic subsets are shown in each graph. ( n = 7 for each group). F) Relative fluorescence of Ki‐67 in CD4 + CD8 + double positive (DP) T cells under anti‐CD3/CD28 stimulation. Total thymocytes from Ripk3 +/+ ( n = 5) and Ripk3 −/− ( n = 5) mice were stimulated with anti‐CD3 (1 µg mL −1 ) and anti‐CD28 (2 µg mL −1 ) or without, and then Ki‐67 in DP cell was measured by FACS. Statistical analyses were performed using the two‐tailed unpaired Student t ‐test. P values below 0.05 were considered significant in the following manner: * p < 0.05, ** p < 0.01, *** p < 0.001.

    Journal: Advanced Science

    Article Title: LCK‐Mediated RIPK3 Activation Controls Double‐Positive Thymocyte Proliferation and Restrains Thymic Lymphoma by Regulating the PP2A‐ERK Axis

    doi: 10.1002/advs.202204522

    Figure Lengend Snippet: Genetic loss of RIPK3 leads to hyperproliferation of DP thymocytes contributing to thymic lymphoma. A) Kaplan–Meier survival curve (Long‐rank test) in Ripk3 +/+ ( n = 51) and Ripk3 −/− ( n = 40) mice. B) Spontaneous tumors found in various organs and their frequency in Ripk3 +/+ and Ripk3 −/− animals. C) Thymus from Ripk3 +/+ and Ripk3 −/− was shown (left panel) and its weight was shown in the graph (right panel). ( n = 13 for each group). D) Representative H&E images of thymus from both Ripk3 +/+ and Ripk3 −/− mice. E) Percentage of the thymic T cell subsets (left panel) and the absolute cell number (right panel) by the indicated thymic subsets are shown in each graph. ( n = 7 for each group). F) Relative fluorescence of Ki‐67 in CD4 + CD8 + double positive (DP) T cells under anti‐CD3/CD28 stimulation. Total thymocytes from Ripk3 +/+ ( n = 5) and Ripk3 −/− ( n = 5) mice were stimulated with anti‐CD3 (1 µg mL −1 ) and anti‐CD28 (2 µg mL −1 ) or without, and then Ki‐67 in DP cell was measured by FACS. Statistical analyses were performed using the two‐tailed unpaired Student t ‐test. P values below 0.05 were considered significant in the following manner: * p < 0.05, ** p < 0.01, *** p < 0.001.

    Article Snippet: Primary antibodies: anti‐RIPK1 (610 459, BD Bioscience), anti‐Mouse‐RIPK3 (ADI‐905‐242‐100, Enzo), anti‐Human‐RIPK3 (13 526, Cell Signaling Technology), anti‐Mouse‐p‐RIPK3 (91 702, Cell Signaling Technology), anti‐Human‐p‐RIPK3 (ab209384, Abcam), anti‐MLKL (ab184718, Abcam), anti‐GAPDH (25 778, Santa Cruz Biotechnology), anti‐ACTIN (sc4778, Santa Cruz Biotechnology), anti‐VINCULIN (V9131, Sigma–Aldrich), anti‐ERK (9102, Cell Signaling Technology), anti‐p‐ERK (9101, Cell Signaling Technology), anti‐LCK (sc‐433, Santa Cruz Biotechnology), anti‐p‐LCK (Y393) (ab208787, Abcam), anti‐p‐Tyr (9411, Cell Signaling Technology), anti‐p‐Tyr (8954, Cell Signaling Technology), anti‐PPP2R2A (5689, Cell Signaling Technology), anti‐PPP2CA (610 555, BD Bioscience), anti‐GST (27‐4577, GE Healthcare) and anti‐FLAG (F1804, Sigma–Aldrich).

    Techniques: Fluorescence, Two Tailed Test

    RIPK3‐MLKL axis does not impact the proliferation and death of DP thymocytes. A) Total thymocytes from Ripk3 +/+ ( n = 6) and Ripk3 −/− ( n = 6) mice were treated with TNF‐ α (50 ng mL −1 ), z‐VAD (20 µ m ) and Smac mimetic (200 n m ) for 5 and 24 h. Dead cells were identified by Annexin V and PI staining. B) The expression level of MLKL protein in primary immune cells. MLKL expression was measured by Western blot analysis. C) Representative histograms were the MLKL expression of the indicated thymic and splenic subsets. Expression levels of MLKL were measured by flow cytometry. Total thymocytes and splenocytes were treated with TNF‐ α (50 ng mL −1 ), z‐VAD (20 µ m ) and Smac mimetic (200 n m ) for the indicated time or without, and cells were analyzed by Annexin V and Propidium Iodide (PI) staining and measured by D) Flow Cytometry. RIPK3, MLKL and p ‐MLKL protein was measured by E) Western blot analysis. Statistical analyses were performed using the two‐tailed unpaired Student t ‐test. P values below 0.05 were considered significant in the following manner: * p < 0.05, ** p < 0.01, *** p < 0.001.

    Journal: Advanced Science

    Article Title: LCK‐Mediated RIPK3 Activation Controls Double‐Positive Thymocyte Proliferation and Restrains Thymic Lymphoma by Regulating the PP2A‐ERK Axis

    doi: 10.1002/advs.202204522

    Figure Lengend Snippet: RIPK3‐MLKL axis does not impact the proliferation and death of DP thymocytes. A) Total thymocytes from Ripk3 +/+ ( n = 6) and Ripk3 −/− ( n = 6) mice were treated with TNF‐ α (50 ng mL −1 ), z‐VAD (20 µ m ) and Smac mimetic (200 n m ) for 5 and 24 h. Dead cells were identified by Annexin V and PI staining. B) The expression level of MLKL protein in primary immune cells. MLKL expression was measured by Western blot analysis. C) Representative histograms were the MLKL expression of the indicated thymic and splenic subsets. Expression levels of MLKL were measured by flow cytometry. Total thymocytes and splenocytes were treated with TNF‐ α (50 ng mL −1 ), z‐VAD (20 µ m ) and Smac mimetic (200 n m ) for the indicated time or without, and cells were analyzed by Annexin V and Propidium Iodide (PI) staining and measured by D) Flow Cytometry. RIPK3, MLKL and p ‐MLKL protein was measured by E) Western blot analysis. Statistical analyses were performed using the two‐tailed unpaired Student t ‐test. P values below 0.05 were considered significant in the following manner: * p < 0.05, ** p < 0.01, *** p < 0.001.

    Article Snippet: Primary antibodies: anti‐RIPK1 (610 459, BD Bioscience), anti‐Mouse‐RIPK3 (ADI‐905‐242‐100, Enzo), anti‐Human‐RIPK3 (13 526, Cell Signaling Technology), anti‐Mouse‐p‐RIPK3 (91 702, Cell Signaling Technology), anti‐Human‐p‐RIPK3 (ab209384, Abcam), anti‐MLKL (ab184718, Abcam), anti‐GAPDH (25 778, Santa Cruz Biotechnology), anti‐ACTIN (sc4778, Santa Cruz Biotechnology), anti‐VINCULIN (V9131, Sigma–Aldrich), anti‐ERK (9102, Cell Signaling Technology), anti‐p‐ERK (9101, Cell Signaling Technology), anti‐LCK (sc‐433, Santa Cruz Biotechnology), anti‐p‐LCK (Y393) (ab208787, Abcam), anti‐p‐Tyr (9411, Cell Signaling Technology), anti‐p‐Tyr (8954, Cell Signaling Technology), anti‐PPP2R2A (5689, Cell Signaling Technology), anti‐PPP2CA (610 555, BD Bioscience), anti‐GST (27‐4577, GE Healthcare) and anti‐FLAG (F1804, Sigma–Aldrich).

    Techniques: Staining, Expressing, Western Blot, Flow Cytometry, Two Tailed Test

    Carcinogen‐induced thymic tumorigenesis was accelerated in RIPK3‐deficient mice. A) Intraperitoneal injection of ENU ( N ‐ethyl‐ N ‐nitrosourea) at around postnatal day 13 ∼15 (three‐time; 13∼15 day) was performed to both Ripk3 +/+ and Ripk3 −/− mice. B) Kaplan–Meier survival curve (Long‐rank test) after 3 consecutive daily ENU injections into Ripk3 wildtype and Ripk3 knockout mice ( n = 10 for each group). Around 105 days after ENU injection, 50% of Ripk3 −/− mice succumbed to tumors (red dotted line). C) All of animals were examined at 100 days after 3 consecutive daily ENU injection to Ripk3 +/+ ( n = 6) and Ripk3 −/− ( n = 6) mice. The summary of tumor incidence in this ENU injection experiment. 83% of the Ripk3 −/‐ animals developed thymic lymphoma at the microscopic level. D) Schematic diagram of an ENU injection experiment. E) Gross view of the abnormally big thymus removed from ENU injected Ripk3 +/+ and Ripk3 −/− mice at day 60. Ripk3 −/− thymus showed a more hyperplasia than Ripk3 +/+ thymus. F) Representative images of hematoxylin and eosin (H&E) stained sections and Ki‐67 staining of thymus from both ENU injected Ripk3 +/+ and Ripk3 −/− mice. G) Relative fluorescence of Ki‐67 in indicated thymic T cells (blue box in Ripk3 +/+ and red box in Ripk3 −/− ) analyzed by Flow Cytometry ( n = 10 ∼ 12 for each group). Statistical analyses were performed using the two‐tailed unpaired Student t ‐test or log rank test. P values below 0.05 were considered significant in the following manner: * p < 0.05, ** p < 0.01, *** p < 0.001.

    Journal: Advanced Science

    Article Title: LCK‐Mediated RIPK3 Activation Controls Double‐Positive Thymocyte Proliferation and Restrains Thymic Lymphoma by Regulating the PP2A‐ERK Axis

    doi: 10.1002/advs.202204522

    Figure Lengend Snippet: Carcinogen‐induced thymic tumorigenesis was accelerated in RIPK3‐deficient mice. A) Intraperitoneal injection of ENU ( N ‐ethyl‐ N ‐nitrosourea) at around postnatal day 13 ∼15 (three‐time; 13∼15 day) was performed to both Ripk3 +/+ and Ripk3 −/− mice. B) Kaplan–Meier survival curve (Long‐rank test) after 3 consecutive daily ENU injections into Ripk3 wildtype and Ripk3 knockout mice ( n = 10 for each group). Around 105 days after ENU injection, 50% of Ripk3 −/− mice succumbed to tumors (red dotted line). C) All of animals were examined at 100 days after 3 consecutive daily ENU injection to Ripk3 +/+ ( n = 6) and Ripk3 −/− ( n = 6) mice. The summary of tumor incidence in this ENU injection experiment. 83% of the Ripk3 −/‐ animals developed thymic lymphoma at the microscopic level. D) Schematic diagram of an ENU injection experiment. E) Gross view of the abnormally big thymus removed from ENU injected Ripk3 +/+ and Ripk3 −/− mice at day 60. Ripk3 −/− thymus showed a more hyperplasia than Ripk3 +/+ thymus. F) Representative images of hematoxylin and eosin (H&E) stained sections and Ki‐67 staining of thymus from both ENU injected Ripk3 +/+ and Ripk3 −/− mice. G) Relative fluorescence of Ki‐67 in indicated thymic T cells (blue box in Ripk3 +/+ and red box in Ripk3 −/− ) analyzed by Flow Cytometry ( n = 10 ∼ 12 for each group). Statistical analyses were performed using the two‐tailed unpaired Student t ‐test or log rank test. P values below 0.05 were considered significant in the following manner: * p < 0.05, ** p < 0.01, *** p < 0.001.

    Article Snippet: Primary antibodies: anti‐RIPK1 (610 459, BD Bioscience), anti‐Mouse‐RIPK3 (ADI‐905‐242‐100, Enzo), anti‐Human‐RIPK3 (13 526, Cell Signaling Technology), anti‐Mouse‐p‐RIPK3 (91 702, Cell Signaling Technology), anti‐Human‐p‐RIPK3 (ab209384, Abcam), anti‐MLKL (ab184718, Abcam), anti‐GAPDH (25 778, Santa Cruz Biotechnology), anti‐ACTIN (sc4778, Santa Cruz Biotechnology), anti‐VINCULIN (V9131, Sigma–Aldrich), anti‐ERK (9102, Cell Signaling Technology), anti‐p‐ERK (9101, Cell Signaling Technology), anti‐LCK (sc‐433, Santa Cruz Biotechnology), anti‐p‐LCK (Y393) (ab208787, Abcam), anti‐p‐Tyr (9411, Cell Signaling Technology), anti‐p‐Tyr (8954, Cell Signaling Technology), anti‐PPP2R2A (5689, Cell Signaling Technology), anti‐PPP2CA (610 555, BD Bioscience), anti‐GST (27‐4577, GE Healthcare) and anti‐FLAG (F1804, Sigma–Aldrich).

    Techniques: Injection, Knock-Out, Staining, Fluorescence, Flow Cytometry, Two Tailed Test

    RIPK3 deficiency enhances thymic lymphoma in p53‐deficient mice via ERK hyperactivation. A) Kaplan‐Meier survival curve (Long‐rank test) of animals in different genetic backgrounds including Ripk3 +/+ p53 −/− and Ripk3 −/− p53 −/− animals. Red dotted line shows median survival of Ripk3 +/+ p53 −/− and Ripk3 −/− p53 −/‐ mice, which are significantly different ( p < 0.0001). B) The thymic lymphoma free survival curve and thymic lymphoma incidence of p53 −/− and Ripk3 −/− p53 −/− animals until 150 days of age. The tumor‐free survival plot of Ripk3 +/+ p53 −/− ( n = 42) compared with Ripk3 −/− p53 −/‐ ( n = 158) mice that are significantly different. C) Representative images of thymus, spleen and lymph node from tumor‐bearing Ripk3 +/+ p53 −/− and Ripk3 −/− p53 −/− littermates. D) The phenotype of the thymic T cell populations (left panel). The thymic T cell from both Ripk3 +/+ p53 −/− and Ripk3 −/− p53 −/− thymic lymphoma showed homogenous expansion in DP T cells but Ripk3 −/− p53 −/− DP T cells showed increased proliferation index (Ki‐67) (right panel). E) RNA sequencing analysis of up‐ and down‐regulated gene in Ripk3 −/− p53 −/− thymic lymphoma compared to p53 −/− thymic lymphoma. Analysis of up‐regulated gene shown the change in positive regulation of ERK signaling pathways. F) Total thymocytes from Ripk3 +/+ ( n = 4) and Ripk3 −/− ( n = 4) mice were stimulated with anti‐CD3 (1 µg mL −1 ) and anti‐CD28 (2 µg mL −1 ) or without for indicated time. Phosphorylation of ERK in DP T cell measured by Flow Cytometry. G) The protein expression levels of p‐ERK in normal thymus ( Ripk3 +/− p53 +/− ; n = 3 , Ripk3 −/− p53 +/+ ; n = 3) and thymic lymphoma ( Ripk3 +/+ p53 −/− ; n = 10 , Ripk3 −/− p53 −/− ; n = 10) tissues were measured by Western blot analysis. Statistical analyses were performed using the two‐tailed unpaired Student t ‐test or log rank test. p values below 0.05 were considered significant in the following manner: * p < 0.05, ** p < 0.01, *** p < 0.001.

    Journal: Advanced Science

    Article Title: LCK‐Mediated RIPK3 Activation Controls Double‐Positive Thymocyte Proliferation and Restrains Thymic Lymphoma by Regulating the PP2A‐ERK Axis

    doi: 10.1002/advs.202204522

    Figure Lengend Snippet: RIPK3 deficiency enhances thymic lymphoma in p53‐deficient mice via ERK hyperactivation. A) Kaplan‐Meier survival curve (Long‐rank test) of animals in different genetic backgrounds including Ripk3 +/+ p53 −/− and Ripk3 −/− p53 −/− animals. Red dotted line shows median survival of Ripk3 +/+ p53 −/− and Ripk3 −/− p53 −/‐ mice, which are significantly different ( p < 0.0001). B) The thymic lymphoma free survival curve and thymic lymphoma incidence of p53 −/− and Ripk3 −/− p53 −/− animals until 150 days of age. The tumor‐free survival plot of Ripk3 +/+ p53 −/− ( n = 42) compared with Ripk3 −/− p53 −/‐ ( n = 158) mice that are significantly different. C) Representative images of thymus, spleen and lymph node from tumor‐bearing Ripk3 +/+ p53 −/− and Ripk3 −/− p53 −/− littermates. D) The phenotype of the thymic T cell populations (left panel). The thymic T cell from both Ripk3 +/+ p53 −/− and Ripk3 −/− p53 −/− thymic lymphoma showed homogenous expansion in DP T cells but Ripk3 −/− p53 −/− DP T cells showed increased proliferation index (Ki‐67) (right panel). E) RNA sequencing analysis of up‐ and down‐regulated gene in Ripk3 −/− p53 −/− thymic lymphoma compared to p53 −/− thymic lymphoma. Analysis of up‐regulated gene shown the change in positive regulation of ERK signaling pathways. F) Total thymocytes from Ripk3 +/+ ( n = 4) and Ripk3 −/− ( n = 4) mice were stimulated with anti‐CD3 (1 µg mL −1 ) and anti‐CD28 (2 µg mL −1 ) or without for indicated time. Phosphorylation of ERK in DP T cell measured by Flow Cytometry. G) The protein expression levels of p‐ERK in normal thymus ( Ripk3 +/− p53 +/− ; n = 3 , Ripk3 −/− p53 +/+ ; n = 3) and thymic lymphoma ( Ripk3 +/+ p53 −/− ; n = 10 , Ripk3 −/− p53 −/− ; n = 10) tissues were measured by Western blot analysis. Statistical analyses were performed using the two‐tailed unpaired Student t ‐test or log rank test. p values below 0.05 were considered significant in the following manner: * p < 0.05, ** p < 0.01, *** p < 0.001.

    Article Snippet: Primary antibodies: anti‐RIPK1 (610 459, BD Bioscience), anti‐Mouse‐RIPK3 (ADI‐905‐242‐100, Enzo), anti‐Human‐RIPK3 (13 526, Cell Signaling Technology), anti‐Mouse‐p‐RIPK3 (91 702, Cell Signaling Technology), anti‐Human‐p‐RIPK3 (ab209384, Abcam), anti‐MLKL (ab184718, Abcam), anti‐GAPDH (25 778, Santa Cruz Biotechnology), anti‐ACTIN (sc4778, Santa Cruz Biotechnology), anti‐VINCULIN (V9131, Sigma–Aldrich), anti‐ERK (9102, Cell Signaling Technology), anti‐p‐ERK (9101, Cell Signaling Technology), anti‐LCK (sc‐433, Santa Cruz Biotechnology), anti‐p‐LCK (Y393) (ab208787, Abcam), anti‐p‐Tyr (9411, Cell Signaling Technology), anti‐p‐Tyr (8954, Cell Signaling Technology), anti‐PPP2R2A (5689, Cell Signaling Technology), anti‐PPP2CA (610 555, BD Bioscience), anti‐GST (27‐4577, GE Healthcare) and anti‐FLAG (F1804, Sigma–Aldrich).

    Techniques: RNA Sequencing Assay, Flow Cytometry, Expressing, Western Blot, Two Tailed Test

    TCR‐mediated activation of LCK interacts with and RIPK3 to promote RIPK3 phosphorylation. A) Total thymocytes from Ripk3 +/+ ( n = 5) and Ripk3 −/− ( n = 5) mice were cultured with anti‐CD3 (1 µg mL −1 ) and anti‐CD28 (2 µg mL −1 ) or without for indicated time. Relative fluorescence of RIPK3 and p‐RIPK3 in DP T cells. B) Phosphorylation of RIPK3 increased in anti‐CD3 (1 µg mL −1 ) and anti‐CD28 (2 µg mL −1 ) stimulation. C) Experimental workflow for TAP (Tandem Affinity purification) pull‐down assay. An aliquot of each purified sample was loaded to SDS‐PAGE and stained with Coomassie brilliant blue. Potential RIPK3‐binding proteins were identified by LC‐MS/MS. Venn diagram represents the overlap of proteins and unique proteins identified by LC/MS/MS among TAP‐purified samples as indicated. Total 503 proteins were identified as specific RIPK3 binding proteins. There were several kinases and phosphatases. D) Computational docking model for RIPK3 (olive) and LCK (blue) predicted using ClusPro (see Materials and Methods). E) Western blot analysis after immunoprecipitation of mouse RIPK3 and mouse LCK in HEK293T cells. HEK293T cells were transfected with LCK and/or Flag‐RIPK3 expression constructs. Cells were harvested at 24 h after transfection. The endogenous RIPK3 interacted with LCK in response to anti‐CD3/CD28 stimulation. Interaction of RIPK3 and LCK was observed by F) Western blot analysis, and confirmed with G) Duolink proximity ligation assay. H) Tyrosine phosphorylation of RIPK3 were detected in LCK and Flag‐RIPK3 expressing HEK293T cells. Statistical analyses were performed using the two‐tailed unpaired Student t ‐test. p values below 0.05 were considered significant in the following manner: * p < 0.05, ** p < 0.01, *** p < 0.001.

    Journal: Advanced Science

    Article Title: LCK‐Mediated RIPK3 Activation Controls Double‐Positive Thymocyte Proliferation and Restrains Thymic Lymphoma by Regulating the PP2A‐ERK Axis

    doi: 10.1002/advs.202204522

    Figure Lengend Snippet: TCR‐mediated activation of LCK interacts with and RIPK3 to promote RIPK3 phosphorylation. A) Total thymocytes from Ripk3 +/+ ( n = 5) and Ripk3 −/− ( n = 5) mice were cultured with anti‐CD3 (1 µg mL −1 ) and anti‐CD28 (2 µg mL −1 ) or without for indicated time. Relative fluorescence of RIPK3 and p‐RIPK3 in DP T cells. B) Phosphorylation of RIPK3 increased in anti‐CD3 (1 µg mL −1 ) and anti‐CD28 (2 µg mL −1 ) stimulation. C) Experimental workflow for TAP (Tandem Affinity purification) pull‐down assay. An aliquot of each purified sample was loaded to SDS‐PAGE and stained with Coomassie brilliant blue. Potential RIPK3‐binding proteins were identified by LC‐MS/MS. Venn diagram represents the overlap of proteins and unique proteins identified by LC/MS/MS among TAP‐purified samples as indicated. Total 503 proteins were identified as specific RIPK3 binding proteins. There were several kinases and phosphatases. D) Computational docking model for RIPK3 (olive) and LCK (blue) predicted using ClusPro (see Materials and Methods). E) Western blot analysis after immunoprecipitation of mouse RIPK3 and mouse LCK in HEK293T cells. HEK293T cells were transfected with LCK and/or Flag‐RIPK3 expression constructs. Cells were harvested at 24 h after transfection. The endogenous RIPK3 interacted with LCK in response to anti‐CD3/CD28 stimulation. Interaction of RIPK3 and LCK was observed by F) Western blot analysis, and confirmed with G) Duolink proximity ligation assay. H) Tyrosine phosphorylation of RIPK3 were detected in LCK and Flag‐RIPK3 expressing HEK293T cells. Statistical analyses were performed using the two‐tailed unpaired Student t ‐test. p values below 0.05 were considered significant in the following manner: * p < 0.05, ** p < 0.01, *** p < 0.001.

    Article Snippet: Primary antibodies: anti‐RIPK1 (610 459, BD Bioscience), anti‐Mouse‐RIPK3 (ADI‐905‐242‐100, Enzo), anti‐Human‐RIPK3 (13 526, Cell Signaling Technology), anti‐Mouse‐p‐RIPK3 (91 702, Cell Signaling Technology), anti‐Human‐p‐RIPK3 (ab209384, Abcam), anti‐MLKL (ab184718, Abcam), anti‐GAPDH (25 778, Santa Cruz Biotechnology), anti‐ACTIN (sc4778, Santa Cruz Biotechnology), anti‐VINCULIN (V9131, Sigma–Aldrich), anti‐ERK (9102, Cell Signaling Technology), anti‐p‐ERK (9101, Cell Signaling Technology), anti‐LCK (sc‐433, Santa Cruz Biotechnology), anti‐p‐LCK (Y393) (ab208787, Abcam), anti‐p‐Tyr (9411, Cell Signaling Technology), anti‐p‐Tyr (8954, Cell Signaling Technology), anti‐PPP2R2A (5689, Cell Signaling Technology), anti‐PPP2CA (610 555, BD Bioscience), anti‐GST (27‐4577, GE Healthcare) and anti‐FLAG (F1804, Sigma–Aldrich).

    Techniques: Activation Assay, Cell Culture, Fluorescence, Affinity Purification, Pull Down Assay, Purification, SDS Page, Staining, Binding Assay, Liquid Chromatography with Mass Spectroscopy, Western Blot, Immunoprecipitation, Transfection, Expressing, Construct, Proximity Ligation Assay, Two Tailed Test

    RIPK3 suppresses ERK phosphorylation by activating PP2A function. A) Western blot analysis of ERK phosphorylation after transiently expression of human RIPK3 and human LCK in HEK293T cells. HEK293T cells were transfected with GST‐LCK and/or Flag‐RIPK3 expression constructs. B) Computational docking model for RIPK3 (olive) and PP2A predicted using ClusPro. The PP2A comprises three subunits (A subunit; blue, B55 α subunit; green, and C subunit: magenta). C) Western blot analysis after immunoprecipitation of human RIPK3 and human PP2A subunits in HEK293T cells. HEK293T cells were transfected with GFP‐PPP2CA or GFP‐PPP2R2A and/or Flag‐RIPK3 expression constructs. Cells were harvested at 24 h after transfection. D) The endogenous RIPK3 interacted with PPP2R2A in response to anti‐CD3/CD28 stimulation. E) In vitro kinase activity of RIPK3 toward PPP2R2A with 32 P‐labeled ATP. To avoid false positive artifacts in the in vitro kinase assay, we included conditions with or without ATP. GST‐RIPK3 (amino acid 2–328) and GST‐PPP2R2A recombinant proteins were purified from sf‐9 cells. F) Total thymocytes from Ripk3 +/+ and Ripk3 −/− mice were stimulated with anti‐CD3 (1 µg mL −1 ) and anti‐CD28 (2 µg mL −1 ) for indicated time. Cell lysates were immunoprecipitated with anti‐ERK antibody. Immunocomplexes and cell lysates were analyzed by immunoblotting.

    Journal: Advanced Science

    Article Title: LCK‐Mediated RIPK3 Activation Controls Double‐Positive Thymocyte Proliferation and Restrains Thymic Lymphoma by Regulating the PP2A‐ERK Axis

    doi: 10.1002/advs.202204522

    Figure Lengend Snippet: RIPK3 suppresses ERK phosphorylation by activating PP2A function. A) Western blot analysis of ERK phosphorylation after transiently expression of human RIPK3 and human LCK in HEK293T cells. HEK293T cells were transfected with GST‐LCK and/or Flag‐RIPK3 expression constructs. B) Computational docking model for RIPK3 (olive) and PP2A predicted using ClusPro. The PP2A comprises three subunits (A subunit; blue, B55 α subunit; green, and C subunit: magenta). C) Western blot analysis after immunoprecipitation of human RIPK3 and human PP2A subunits in HEK293T cells. HEK293T cells were transfected with GFP‐PPP2CA or GFP‐PPP2R2A and/or Flag‐RIPK3 expression constructs. Cells were harvested at 24 h after transfection. D) The endogenous RIPK3 interacted with PPP2R2A in response to anti‐CD3/CD28 stimulation. E) In vitro kinase activity of RIPK3 toward PPP2R2A with 32 P‐labeled ATP. To avoid false positive artifacts in the in vitro kinase assay, we included conditions with or without ATP. GST‐RIPK3 (amino acid 2–328) and GST‐PPP2R2A recombinant proteins were purified from sf‐9 cells. F) Total thymocytes from Ripk3 +/+ and Ripk3 −/− mice were stimulated with anti‐CD3 (1 µg mL −1 ) and anti‐CD28 (2 µg mL −1 ) for indicated time. Cell lysates were immunoprecipitated with anti‐ERK antibody. Immunocomplexes and cell lysates were analyzed by immunoblotting.

    Article Snippet: Primary antibodies: anti‐RIPK1 (610 459, BD Bioscience), anti‐Mouse‐RIPK3 (ADI‐905‐242‐100, Enzo), anti‐Human‐RIPK3 (13 526, Cell Signaling Technology), anti‐Mouse‐p‐RIPK3 (91 702, Cell Signaling Technology), anti‐Human‐p‐RIPK3 (ab209384, Abcam), anti‐MLKL (ab184718, Abcam), anti‐GAPDH (25 778, Santa Cruz Biotechnology), anti‐ACTIN (sc4778, Santa Cruz Biotechnology), anti‐VINCULIN (V9131, Sigma–Aldrich), anti‐ERK (9102, Cell Signaling Technology), anti‐p‐ERK (9101, Cell Signaling Technology), anti‐LCK (sc‐433, Santa Cruz Biotechnology), anti‐p‐LCK (Y393) (ab208787, Abcam), anti‐p‐Tyr (9411, Cell Signaling Technology), anti‐p‐Tyr (8954, Cell Signaling Technology), anti‐PPP2R2A (5689, Cell Signaling Technology), anti‐PPP2CA (610 555, BD Bioscience), anti‐GST (27‐4577, GE Healthcare) and anti‐FLAG (F1804, Sigma–Aldrich).

    Techniques: Western Blot, Expressing, Transfection, Construct, Immunoprecipitation, In Vitro, Activity Assay, Labeling, Kinase Assay, Recombinant, Purification

    Pharmacological modulation of PP2A activity regulates DP thymocyte hyperproliferation‐associated tumorigenesis. A) Intraperitoneal injection of ENU at around postnatal day 13 ∼15 (three‐time) was performed to Ripk3 +/+ mice. After 30 days of injection, mice were injected with LB‐100 or PBS intraperitoneally at 2 mg kg −1 on alternate days for 30 days. ( n = 4 for each group). B) Phosphorylation of ERK increased in response to anti‐CD3 (1 µg mL −1 ) and anti‐CD28 (2 µg mL −1 ) stimulation, which was further increased by LB‐100 treatment. C) Activation of ERK increased in LB‐100 injected Ripk3 +/+ thymus. D) Relative fluorescence of Ki‐67 in thymic T cells analyzed by Flow Cytometry. Ki‐67 expression is enhanced in LB‐100 injected Ripk3 +/+ thymocytes. E) Schematic diagram of an ENU injection experiment. After 30 days of ENU injection, Ripk3 −/− mice were injected with SMAP or PBS intraperitoneally at 2 mg kg −1 on alternate days for 30 days. ( n = 4 – 6 for each group). F) Activation of ERK reduced in SMAP injected Ripk3 −/− thymus. G) Ki‐67 expression is decreased in SMAP injected Ripk3 −/− thymocytes. Relative fluorescence of Ki‐67 expression analyzed by Flow Cytometry. Statistical analyses were performed using the two‐tailed unpaired Student t ‐test. P values below 0.05 were considered significant in the following manner: * p < 0.05, ** p < 0.01, *** p < 0.001.

    Journal: Advanced Science

    Article Title: LCK‐Mediated RIPK3 Activation Controls Double‐Positive Thymocyte Proliferation and Restrains Thymic Lymphoma by Regulating the PP2A‐ERK Axis

    doi: 10.1002/advs.202204522

    Figure Lengend Snippet: Pharmacological modulation of PP2A activity regulates DP thymocyte hyperproliferation‐associated tumorigenesis. A) Intraperitoneal injection of ENU at around postnatal day 13 ∼15 (three‐time) was performed to Ripk3 +/+ mice. After 30 days of injection, mice were injected with LB‐100 or PBS intraperitoneally at 2 mg kg −1 on alternate days for 30 days. ( n = 4 for each group). B) Phosphorylation of ERK increased in response to anti‐CD3 (1 µg mL −1 ) and anti‐CD28 (2 µg mL −1 ) stimulation, which was further increased by LB‐100 treatment. C) Activation of ERK increased in LB‐100 injected Ripk3 +/+ thymus. D) Relative fluorescence of Ki‐67 in thymic T cells analyzed by Flow Cytometry. Ki‐67 expression is enhanced in LB‐100 injected Ripk3 +/+ thymocytes. E) Schematic diagram of an ENU injection experiment. After 30 days of ENU injection, Ripk3 −/− mice were injected with SMAP or PBS intraperitoneally at 2 mg kg −1 on alternate days for 30 days. ( n = 4 – 6 for each group). F) Activation of ERK reduced in SMAP injected Ripk3 −/− thymus. G) Ki‐67 expression is decreased in SMAP injected Ripk3 −/− thymocytes. Relative fluorescence of Ki‐67 expression analyzed by Flow Cytometry. Statistical analyses were performed using the two‐tailed unpaired Student t ‐test. P values below 0.05 were considered significant in the following manner: * p < 0.05, ** p < 0.01, *** p < 0.001.

    Article Snippet: Primary antibodies: anti‐RIPK1 (610 459, BD Bioscience), anti‐Mouse‐RIPK3 (ADI‐905‐242‐100, Enzo), anti‐Human‐RIPK3 (13 526, Cell Signaling Technology), anti‐Mouse‐p‐RIPK3 (91 702, Cell Signaling Technology), anti‐Human‐p‐RIPK3 (ab209384, Abcam), anti‐MLKL (ab184718, Abcam), anti‐GAPDH (25 778, Santa Cruz Biotechnology), anti‐ACTIN (sc4778, Santa Cruz Biotechnology), anti‐VINCULIN (V9131, Sigma–Aldrich), anti‐ERK (9102, Cell Signaling Technology), anti‐p‐ERK (9101, Cell Signaling Technology), anti‐LCK (sc‐433, Santa Cruz Biotechnology), anti‐p‐LCK (Y393) (ab208787, Abcam), anti‐p‐Tyr (9411, Cell Signaling Technology), anti‐p‐Tyr (8954, Cell Signaling Technology), anti‐PPP2R2A (5689, Cell Signaling Technology), anti‐PPP2CA (610 555, BD Bioscience), anti‐GST (27‐4577, GE Healthcare) and anti‐FLAG (F1804, Sigma–Aldrich).

    Techniques: Activity Assay, Injection, Activation Assay, Fluorescence, Flow Cytometry, Expressing, Two Tailed Test

    (A) Representative immunoblot images of RIPK3 phosphorylation (Thr231/Ser232), RIPK3, MLKL phosphorylation (Ser345), MLKL, RIPK1 phosphorylation (Ser166) protein and (B) quantification of RIPK3 phosphorylation, RIPK3, MLKL phosphorylation, RIPK1 phosphorylation protein levels in lung tissues from mice treated with oligomycin either control or LPS (n=3 per each group). β-actin or MLKL were used as loading control. Data are representative of three independent experiments. Data are mean±SD. * p<0.05; ** p<0.01; *** p<0.001 by Student’s 2-tailed t-test or ANOVA.

    Journal: Immune Network

    Article Title: Impairment of Mitochondrial ATP Synthesis Induces RIPK3-dependent Necroptosis in Lung Epithelial Cells During Lung Injury by Lung Inflammation

    doi: 10.4110/in.2022.22.e18

    Figure Lengend Snippet: (A) Representative immunoblot images of RIPK3 phosphorylation (Thr231/Ser232), RIPK3, MLKL phosphorylation (Ser345), MLKL, RIPK1 phosphorylation (Ser166) protein and (B) quantification of RIPK3 phosphorylation, RIPK3, MLKL phosphorylation, RIPK1 phosphorylation protein levels in lung tissues from mice treated with oligomycin either control or LPS (n=3 per each group). β-actin or MLKL were used as loading control. Data are representative of three independent experiments. Data are mean±SD. * p<0.05; ** p<0.01; *** p<0.001 by Student’s 2-tailed t-test or ANOVA.

    Article Snippet: After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA) or Protran nitrocellulose membranes (10600001; GE Healthcare Life Science, Pittsburgh, PA, USA) and blocked in 5% non-fat milk or 5% (w/v) BSA (9048-46-8; Santa Cruz Biotechnology, Dallas, TX, USA) in Tris-buffered saline containing 0.05% Tween-20 (TBS-T) (TBS [170-6435; Bio-Rad Laboratories, Hercules, CA, USA] and Tween-20 [170-6531'; Bio-Rad Laboratories]) at 25°C for 1 h. The membranes were immunoblotted with primary Abs against RIPK3 for mouse (AHP1797; Bio-Rad Laboratories), RIPK3 for human (ab62344; Abcam, Cambridge, UK), Phospho-RIPK3 (Thr231/Ser232) for mouse (ab222320; Abcam), Phospho-RIPK3 (Ser227) for human (ab209384; Abcam), Phospho-MLKL (Ser345) (ab196436; Abcam) for mouse, Phospho-MLKL (Ser358) (ab187091; Abcam) for human, MLKL (ab243142; Abcam) for mouse, MLKL (ab184718; Abcam) for human, Phospho-RIPK1 (Ser166) (#31122; Cell Signaling Technology, Danvers, MA, USA) for mouse, Phospho-RIPK1 (Ser166) (NBP3-06877; Novus, Littleton, CO, USA) for human, and β-actin (A5316; Sigma-Aldrich) overnight at 4°C and washed five times with TBS-T for 10 min each at room temperature.

    Techniques: Western Blot

    (A) Representative immunofluorescence images for MLKL phosphorylation at Ser345 (P-MLKL) staining (green) in lung epithelial cells expressing E-cadherin (red) of mice treated with oligomycin either control or LPS. Scale bars, 20 µm. DAPI-stained nuclei are shown in blue. White arrows indicate P-MLKL positive cells (n=10 images per individual subject, n=5 per each group). (B) Quantification of intensity for P-MLKL-positive staining and (C) quantification of P-MLKL and E-cadherin-positive cells from immunofluorescence images in A. (D) Representative immunoblot images of RIPK3 phosphorylation (Thr231/Ser232), RIPK3, MLKL phosphorylation (Ser345), MLKL, RIPK1 phosphorylation (Ser166) protein from lung tissue sections of A. MLKL was used as loading control. (E) Quantification of protein levels in BALF of lungs from mice treated with oligomycin either control or LPS (n=5 per each group). (F) Quantification of total inflammatory cells in BALF of lungs from mice treated with oligomycin either control or LPS (n=5 per each group). Data are representative of three independent experiments. Data are mean±SD. * p<0.05; ** p<0.01; *** p<0.001 by Student’s 2-tailed t-test or ANOVA.

    Journal: Immune Network

    Article Title: Impairment of Mitochondrial ATP Synthesis Induces RIPK3-dependent Necroptosis in Lung Epithelial Cells During Lung Injury by Lung Inflammation

    doi: 10.4110/in.2022.22.e18

    Figure Lengend Snippet: (A) Representative immunofluorescence images for MLKL phosphorylation at Ser345 (P-MLKL) staining (green) in lung epithelial cells expressing E-cadherin (red) of mice treated with oligomycin either control or LPS. Scale bars, 20 µm. DAPI-stained nuclei are shown in blue. White arrows indicate P-MLKL positive cells (n=10 images per individual subject, n=5 per each group). (B) Quantification of intensity for P-MLKL-positive staining and (C) quantification of P-MLKL and E-cadherin-positive cells from immunofluorescence images in A. (D) Representative immunoblot images of RIPK3 phosphorylation (Thr231/Ser232), RIPK3, MLKL phosphorylation (Ser345), MLKL, RIPK1 phosphorylation (Ser166) protein from lung tissue sections of A. MLKL was used as loading control. (E) Quantification of protein levels in BALF of lungs from mice treated with oligomycin either control or LPS (n=5 per each group). (F) Quantification of total inflammatory cells in BALF of lungs from mice treated with oligomycin either control or LPS (n=5 per each group). Data are representative of three independent experiments. Data are mean±SD. * p<0.05; ** p<0.01; *** p<0.001 by Student’s 2-tailed t-test or ANOVA.

    Article Snippet: After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA) or Protran nitrocellulose membranes (10600001; GE Healthcare Life Science, Pittsburgh, PA, USA) and blocked in 5% non-fat milk or 5% (w/v) BSA (9048-46-8; Santa Cruz Biotechnology, Dallas, TX, USA) in Tris-buffered saline containing 0.05% Tween-20 (TBS-T) (TBS [170-6435; Bio-Rad Laboratories, Hercules, CA, USA] and Tween-20 [170-6531'; Bio-Rad Laboratories]) at 25°C for 1 h. The membranes were immunoblotted with primary Abs against RIPK3 for mouse (AHP1797; Bio-Rad Laboratories), RIPK3 for human (ab62344; Abcam, Cambridge, UK), Phospho-RIPK3 (Thr231/Ser232) for mouse (ab222320; Abcam), Phospho-RIPK3 (Ser227) for human (ab209384; Abcam), Phospho-MLKL (Ser345) (ab196436; Abcam) for mouse, Phospho-MLKL (Ser358) (ab187091; Abcam) for human, MLKL (ab243142; Abcam) for mouse, MLKL (ab184718; Abcam) for human, Phospho-RIPK1 (Ser166) (#31122; Cell Signaling Technology, Danvers, MA, USA) for mouse, Phospho-RIPK1 (Ser166) (NBP3-06877; Novus, Littleton, CO, USA) for human, and β-actin (A5316; Sigma-Aldrich) overnight at 4°C and washed five times with TBS-T for 10 min each at room temperature.

    Techniques: Immunofluorescence, Staining, Expressing, Western Blot

    (A) Representative immunohistochemistry images for RIPK3 staining in lung tissues from patients with ARDS (ARDS) and non-ARDS subjects (Normal). Scale bars, 100 µm. Black arrows indicate RIPK3-positive cells. (B) Quantification of intensity for RIPK3-positive staining in cells and (C) quantification of RIPK3-positive cells from immunohistochemistry images in A (ARDS, n=7; Normal, n=2; n = 10 images per individual subject). (D) Representative immunoblot images of RIPK3 phosphorylation (Ser227), RIPK3, MLKL phosphorylation (Ser358), MLKL, RIPK1 phosphorylation (Ser166) protein (left) and quantification of RIPK3 phosphorylation, RIPK3, MLKL phosphorylation, RIPK1 phosphorylation protein levels (right) in lung tissues from ARDS and Normal (n=2 per each group). β-actin or MLKL were used as loading control. Data are representative of three independent experiments. Data are mean±SD. ** p<0.01; *** p<0.001 by Student’s 2-tailed t-test or Mann–Whitney test.

    Journal: Immune Network

    Article Title: Impairment of Mitochondrial ATP Synthesis Induces RIPK3-dependent Necroptosis in Lung Epithelial Cells During Lung Injury by Lung Inflammation

    doi: 10.4110/in.2022.22.e18

    Figure Lengend Snippet: (A) Representative immunohistochemistry images for RIPK3 staining in lung tissues from patients with ARDS (ARDS) and non-ARDS subjects (Normal). Scale bars, 100 µm. Black arrows indicate RIPK3-positive cells. (B) Quantification of intensity for RIPK3-positive staining in cells and (C) quantification of RIPK3-positive cells from immunohistochemistry images in A (ARDS, n=7; Normal, n=2; n = 10 images per individual subject). (D) Representative immunoblot images of RIPK3 phosphorylation (Ser227), RIPK3, MLKL phosphorylation (Ser358), MLKL, RIPK1 phosphorylation (Ser166) protein (left) and quantification of RIPK3 phosphorylation, RIPK3, MLKL phosphorylation, RIPK1 phosphorylation protein levels (right) in lung tissues from ARDS and Normal (n=2 per each group). β-actin or MLKL were used as loading control. Data are representative of three independent experiments. Data are mean±SD. ** p<0.01; *** p<0.001 by Student’s 2-tailed t-test or Mann–Whitney test.

    Article Snippet: After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA) or Protran nitrocellulose membranes (10600001; GE Healthcare Life Science, Pittsburgh, PA, USA) and blocked in 5% non-fat milk or 5% (w/v) BSA (9048-46-8; Santa Cruz Biotechnology, Dallas, TX, USA) in Tris-buffered saline containing 0.05% Tween-20 (TBS-T) (TBS [170-6435; Bio-Rad Laboratories, Hercules, CA, USA] and Tween-20 [170-6531'; Bio-Rad Laboratories]) at 25°C for 1 h. The membranes were immunoblotted with primary Abs against RIPK3 for mouse (AHP1797; Bio-Rad Laboratories), RIPK3 for human (ab62344; Abcam, Cambridge, UK), Phospho-RIPK3 (Thr231/Ser232) for mouse (ab222320; Abcam), Phospho-RIPK3 (Ser227) for human (ab209384; Abcam), Phospho-MLKL (Ser345) (ab196436; Abcam) for mouse, Phospho-MLKL (Ser358) (ab187091; Abcam) for human, MLKL (ab243142; Abcam) for mouse, MLKL (ab184718; Abcam) for human, Phospho-RIPK1 (Ser166) (#31122; Cell Signaling Technology, Danvers, MA, USA) for mouse, Phospho-RIPK1 (Ser166) (NBP3-06877; Novus, Littleton, CO, USA) for human, and β-actin (A5316; Sigma-Aldrich) overnight at 4°C and washed five times with TBS-T for 10 min each at room temperature.

    Techniques: Immunohistochemistry, Staining, Western Blot, MANN-WHITNEY

    (A) Representative immunofluorescence images for MLKL phosphorylation at Ser358 (P-MLKL) staining (green) in lung epithelial cells expressing E-cadherin (red) of lung tissues from patients with ARDS (ARDS) and non-ARDS subjects (Normal). Scale bars, 20 µm. DAPI-stained nuclei are shown in blue. (B) Quantification of intensity for P-MLKL-positive staining and (C) quantification of P-MLKL and E-cadherin-positive cells from immunofluorescence images in A (ARDS, n=7; Normal, n=2; n=10 images per individual subject). (D) Representative immunoblot images of RIPK3 phosphorylation (Ser227), RIPK3, MLKL phosphorylation (Ser358), MLKL, RIPK1 phosphorylation (Ser166) protein from lung tissue sections of A. MLKL was used as loading control. (E) A schematic diagram to summarize our new findings. Red arrow means an increase and blue arrow means a decrease in the diagram. Data are representative of 3 independent experiments. Data are mean ± SD. ** p<0.01 by Student’s 2-tailed t-test.

    Journal: Immune Network

    Article Title: Impairment of Mitochondrial ATP Synthesis Induces RIPK3-dependent Necroptosis in Lung Epithelial Cells During Lung Injury by Lung Inflammation

    doi: 10.4110/in.2022.22.e18

    Figure Lengend Snippet: (A) Representative immunofluorescence images for MLKL phosphorylation at Ser358 (P-MLKL) staining (green) in lung epithelial cells expressing E-cadherin (red) of lung tissues from patients with ARDS (ARDS) and non-ARDS subjects (Normal). Scale bars, 20 µm. DAPI-stained nuclei are shown in blue. (B) Quantification of intensity for P-MLKL-positive staining and (C) quantification of P-MLKL and E-cadherin-positive cells from immunofluorescence images in A (ARDS, n=7; Normal, n=2; n=10 images per individual subject). (D) Representative immunoblot images of RIPK3 phosphorylation (Ser227), RIPK3, MLKL phosphorylation (Ser358), MLKL, RIPK1 phosphorylation (Ser166) protein from lung tissue sections of A. MLKL was used as loading control. (E) A schematic diagram to summarize our new findings. Red arrow means an increase and blue arrow means a decrease in the diagram. Data are representative of 3 independent experiments. Data are mean ± SD. ** p<0.01 by Student’s 2-tailed t-test.

    Article Snippet: After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA) or Protran nitrocellulose membranes (10600001; GE Healthcare Life Science, Pittsburgh, PA, USA) and blocked in 5% non-fat milk or 5% (w/v) BSA (9048-46-8; Santa Cruz Biotechnology, Dallas, TX, USA) in Tris-buffered saline containing 0.05% Tween-20 (TBS-T) (TBS [170-6435; Bio-Rad Laboratories, Hercules, CA, USA] and Tween-20 [170-6531'; Bio-Rad Laboratories]) at 25°C for 1 h. The membranes were immunoblotted with primary Abs against RIPK3 for mouse (AHP1797; Bio-Rad Laboratories), RIPK3 for human (ab62344; Abcam, Cambridge, UK), Phospho-RIPK3 (Thr231/Ser232) for mouse (ab222320; Abcam), Phospho-RIPK3 (Ser227) for human (ab209384; Abcam), Phospho-MLKL (Ser345) (ab196436; Abcam) for mouse, Phospho-MLKL (Ser358) (ab187091; Abcam) for human, MLKL (ab243142; Abcam) for mouse, MLKL (ab184718; Abcam) for human, Phospho-RIPK1 (Ser166) (#31122; Cell Signaling Technology, Danvers, MA, USA) for mouse, Phospho-RIPK1 (Ser166) (NBP3-06877; Novus, Littleton, CO, USA) for human, and β-actin (A5316; Sigma-Aldrich) overnight at 4°C and washed five times with TBS-T for 10 min each at room temperature.

    Techniques: Immunofluorescence, Staining, Expressing, Western Blot

    Immunohistochemical examination for proliferation rate (labelled by PCNA-positive cells), RIPK3- and Phospho-RIPK3 positive cells in the gastric fundus of FVB/N mice at an age of 6 months and gastric body of human stomach. In the stomach of FVB/N mice at the age of 6 months, image ( A ) showed a higher rate of RIPK3-positive cells on fundus glands (arrow in A ) and some on surface mucous cells (arrowhead in A ). B showed that RIPK3-positive cells were mostly located in in the fundus glands and distributed in the isthmus and neck regions (arrow in B ), and few in the base regions. C showed positive cells for active form of RIPK3 (phosphor-RIPK3) in a low density in the mouse gastric glandular cells. In the body of human stomach, image ( D ) visualized that PCNA-positive cells were observed in both surface mucous cells (arrowhead in D ) and gastric glands (arrow in D ). RIPK3-positive cells were also shown in the same compartments of gastric body ( E ). However, RIPK3-positive cells were evenly distributed in the glands. F was the negative isotopic-matched control. ( A – F , IHC images. Counterstained with Hematoxylin, original magnification 400 ×)

    Journal: Journal of Molecular Histology

    Article Title: The expression of RIPK3 is associated with cell turnover of gastric mucosa in the mouse and human stomach

    doi: 10.1007/s10735-021-10001-5

    Figure Lengend Snippet: Immunohistochemical examination for proliferation rate (labelled by PCNA-positive cells), RIPK3- and Phospho-RIPK3 positive cells in the gastric fundus of FVB/N mice at an age of 6 months and gastric body of human stomach. In the stomach of FVB/N mice at the age of 6 months, image ( A ) showed a higher rate of RIPK3-positive cells on fundus glands (arrow in A ) and some on surface mucous cells (arrowhead in A ). B showed that RIPK3-positive cells were mostly located in in the fundus glands and distributed in the isthmus and neck regions (arrow in B ), and few in the base regions. C showed positive cells for active form of RIPK3 (phosphor-RIPK3) in a low density in the mouse gastric glandular cells. In the body of human stomach, image ( D ) visualized that PCNA-positive cells were observed in both surface mucous cells (arrowhead in D ) and gastric glands (arrow in D ). RIPK3-positive cells were also shown in the same compartments of gastric body ( E ). However, RIPK3-positive cells were evenly distributed in the glands. F was the negative isotopic-matched control. ( A – F , IHC images. Counterstained with Hematoxylin, original magnification 400 ×)

    Article Snippet: In addition, to illustrate active RIPK3 form was expressed in mouse gastric mucosal cells, IHC with rabbit anti-mouse phospho-RIPK3 Thr231-Ser232 monoclonal antibody (Abcam, UK) was done in mouse sections.

    Techniques: Immunohistochemical staining

    Counting data of PCNA- and RIPK3-positive cells in the fundus glands of 6-month-old FVB/N mice and body glands of human stomachs. It showed rates of PCNA-positive cell/gland ( White bar) and RIPK3-positive cells/gland ( Grey bar) in the fundus of mice ( A ) and the body of human stomach ( B ) respectively. The number of PCNA-positive cells/gland was higher than that of RIPK3-positive cells/gland and the PCNA/RIPK3 ratio was ~ 1.6 in the fundic glands of mice ( Black bar in A ), and the ratio was ~ 4.7 in the body glands of human ( Black bar in B )

    Journal: Journal of Molecular Histology

    Article Title: The expression of RIPK3 is associated with cell turnover of gastric mucosa in the mouse and human stomach

    doi: 10.1007/s10735-021-10001-5

    Figure Lengend Snippet: Counting data of PCNA- and RIPK3-positive cells in the fundus glands of 6-month-old FVB/N mice and body glands of human stomachs. It showed rates of PCNA-positive cell/gland ( White bar) and RIPK3-positive cells/gland ( Grey bar) in the fundus of mice ( A ) and the body of human stomach ( B ) respectively. The number of PCNA-positive cells/gland was higher than that of RIPK3-positive cells/gland and the PCNA/RIPK3 ratio was ~ 1.6 in the fundic glands of mice ( Black bar in A ), and the ratio was ~ 4.7 in the body glands of human ( Black bar in B )

    Article Snippet: In addition, to illustrate active RIPK3 form was expressed in mouse gastric mucosal cells, IHC with rabbit anti-mouse phospho-RIPK3 Thr231-Ser232 monoclonal antibody (Abcam, UK) was done in mouse sections.

    Techniques:

    Double immunofluorescence examination on the expression of RIPK3 in glandular parietal cells in FVB/N mice and human. In the mouse gastric fundic section, RIPK3 (labelled by FITC, green color in B ) was shown in H + K + -ATPase positive glandular parietal cells (labelled by Alexa-647, red color in A , C for merged RIPK3/H + K + -ATPase). In the human gastric body section, similar colocalization of RIPK3 (labeled by FITC, green color in E ) with H + K + -ATPase-positive glandular parietal cells (labelled by Alexa-647, red color in D ) was observed ( F for merged RIPK3/H + K + -ATPase images). ( A – F , double immunofluorescence staining, original magnification 200×). (Color figure online)

    Journal: Journal of Molecular Histology

    Article Title: The expression of RIPK3 is associated with cell turnover of gastric mucosa in the mouse and human stomach

    doi: 10.1007/s10735-021-10001-5

    Figure Lengend Snippet: Double immunofluorescence examination on the expression of RIPK3 in glandular parietal cells in FVB/N mice and human. In the mouse gastric fundic section, RIPK3 (labelled by FITC, green color in B ) was shown in H + K + -ATPase positive glandular parietal cells (labelled by Alexa-647, red color in A , C for merged RIPK3/H + K + -ATPase). In the human gastric body section, similar colocalization of RIPK3 (labeled by FITC, green color in E ) with H + K + -ATPase-positive glandular parietal cells (labelled by Alexa-647, red color in D ) was observed ( F for merged RIPK3/H + K + -ATPase images). ( A – F , double immunofluorescence staining, original magnification 200×). (Color figure online)

    Article Snippet: In addition, to illustrate active RIPK3 form was expressed in mouse gastric mucosal cells, IHC with rabbit anti-mouse phospho-RIPK3 Thr231-Ser232 monoclonal antibody (Abcam, UK) was done in mouse sections.

    Techniques: Immunofluorescence, Expressing, Labeling, Double Immunofluorescence Staining

    Double immunofluorescence examination of RIPK3-positive cell phenotypes in human gastric lamina propria. Phenotypic analysis revealed that RIPK3-positive cells ( B , E & H ) located in the lamina propria of human stomach were CD3-positive ( A ) lymphocytes (merged image, C ), CD34-positive ( D ) stromal cells (merged image, F ) and SMA-α-positive ( G ) stromal cells (merged image, I ). ( A – L , double immunofluorescence-stained confocal images, original magnification 200×)

    Journal: Journal of Molecular Histology

    Article Title: The expression of RIPK3 is associated with cell turnover of gastric mucosa in the mouse and human stomach

    doi: 10.1007/s10735-021-10001-5

    Figure Lengend Snippet: Double immunofluorescence examination of RIPK3-positive cell phenotypes in human gastric lamina propria. Phenotypic analysis revealed that RIPK3-positive cells ( B , E & H ) located in the lamina propria of human stomach were CD3-positive ( A ) lymphocytes (merged image, C ), CD34-positive ( D ) stromal cells (merged image, F ) and SMA-α-positive ( G ) stromal cells (merged image, I ). ( A – L , double immunofluorescence-stained confocal images, original magnification 200×)

    Article Snippet: In addition, to illustrate active RIPK3 form was expressed in mouse gastric mucosal cells, IHC with rabbit anti-mouse phospho-RIPK3 Thr231-Ser232 monoclonal antibody (Abcam, UK) was done in mouse sections.

    Techniques: Immunofluorescence, Staining