Structured Review

Tocris mk2 inhibitor pf 3644022
Mk2 Inhibitor Pf 3644022, supplied by Tocris, 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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Average 94 stars, based on 1 article reviews
Price from $9.99 to $1999.99
mk2 inhibitor pf 3644022 - by Bioz Stars, 2023-12
94/100 stars

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atp competitive mapkapk2 mk2 inhibitor pf 3644022  (Tocris)

 
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    Structured Review

    Tocris atp competitive mapkapk2 mk2 inhibitor pf 3644022
    Atp Competitive Mapkapk2 Mk2 Inhibitor Pf 3644022, supplied by Tocris, 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/atp competitive mapkapk2 mk2 inhibitor pf 3644022/product/Tocris
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    atp competitive mapkapk2 mk2 inhibitor pf 3644022 - by Bioz Stars, 2023-12
    86/100 stars

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    Structured Review

    Tocris mk2 inhibitor pf
    Mk2 Inhibitor Pf, supplied by Tocris, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mk2 inhibitor pf/product/Tocris
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mk2 inhibitor pf - by Bioz Stars, 2023-12
    94/100 stars

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    Structured Review

    Tocris mk2 inhibitor
    Ripk1 K376R/K376R mutation increases RIPK1 kinase activity. a Immortalized Ripk1 +/+ , Ripk1 K376R/K376R , and Ripk1 −/− MEFs were treated for the indicated time with TNFα (20 ng/ml). The K63-ubiquitylated proteins were isolated by K63-TUBEs and analyzed by western blot. b TNF-RSC was immunoprecipitated using anti-Flag resin in Ripk1 +/+ and Ripk1 K376R/K376R immortalized MEFs treated for the indicated time with Flag-TNFα (100 ng/ml). The immunocomplexes were analyzed by western blot with indicated antibodies. c Nuclear extracts were collected from Ripk1 +/+ , Ripk1 K376R/K376R , and Ripk1 −/− immortalized MEFs treated with TNFα (20 ng/ml) at indicated times and analyzed by western blotting with antibodies against p65 and PCNA. d Ripk1 +/+ and Ripk1 K376R/K376R immortalized MEFs that stably expressed with the Flag-tagged IκBα-SR were stimulated with TNFα (40 ng/ml) for different periods of time. Cell death was measured by SytoxGreen positivity. e , f Complex II was immunoprecipitated using RIPK1 antibody ( e ) or RIPK3 antibody ( f ) in Ripk1 +/+ , Ripk1 K376R/K376R , and Ripk1 −/− immortalized MEFs treated for the indicated time with TSZ ( e ) or TZ ( f ). The immunocomplexes were analyzed by western blotting with indicated antibodies. T, TNFα (20 ng/ml); S, Smac mimetics (1 μM); Z, zVAD.fmk (1 μM). g Ripk1 +/+ and Ripk1 K376R/K376R immortalized MEFs that expressed with the Flag-tagged TAK1/TAB1 or TAK1-∆N were stimulated with TNFα (20 ng/ml) plus zVAD.fmk (10 μM) for different periods of time. The cell lysates were analyzed by western blotting with indicated antibodies. h Cell death of Ripk1 +/+ and Ripk1 K376R/K376R immortalized MEFs treated for 5 h with different stimulators was measured by SytoxGreen positivity. T: TNFα (20 ng/ml), Z: zVAD.fmk (10 μM), TAK1i: TAK1 inhibitor (500 μM), IKKi: IKK inhibitor(5 μM), MK2i: <t>MK2</t> inhibitor (2 μM). In d , h , data are mean ± s.e.m. ( n = 3 independent cell samples for each genotype). Statistical significance was determined using a two-tailed unpaired t test, n.s., P > 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001
    Mk2 Inhibitor, supplied by Tocris, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mk2 inhibitor/product/Tocris
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mk2 inhibitor - by Bioz Stars, 2023-12
    94/100 stars

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    1) Product Images from "K63-linked ubiquitination regulates RIPK1 kinase activity to prevent cell death during embryogenesis and inflammation"

    Article Title: K63-linked ubiquitination regulates RIPK1 kinase activity to prevent cell death during embryogenesis and inflammation

    Journal: Nature Communications

    doi: 10.1038/s41467-019-12033-8

    Ripk1 K376R/K376R mutation increases RIPK1 kinase activity. a Immortalized Ripk1 +/+ , Ripk1 K376R/K376R , and Ripk1 −/− MEFs were treated for the indicated time with TNFα (20 ng/ml). The K63-ubiquitylated proteins were isolated by K63-TUBEs and analyzed by western blot. b TNF-RSC was immunoprecipitated using anti-Flag resin in Ripk1 +/+ and Ripk1 K376R/K376R immortalized MEFs treated for the indicated time with Flag-TNFα (100 ng/ml). The immunocomplexes were analyzed by western blot with indicated antibodies. c Nuclear extracts were collected from Ripk1 +/+ , Ripk1 K376R/K376R , and Ripk1 −/− immortalized MEFs treated with TNFα (20 ng/ml) at indicated times and analyzed by western blotting with antibodies against p65 and PCNA. d Ripk1 +/+ and Ripk1 K376R/K376R immortalized MEFs that stably expressed with the Flag-tagged IκBα-SR were stimulated with TNFα (40 ng/ml) for different periods of time. Cell death was measured by SytoxGreen positivity. e , f Complex II was immunoprecipitated using RIPK1 antibody ( e ) or RIPK3 antibody ( f ) in Ripk1 +/+ , Ripk1 K376R/K376R , and Ripk1 −/− immortalized MEFs treated for the indicated time with TSZ ( e ) or TZ ( f ). The immunocomplexes were analyzed by western blotting with indicated antibodies. T, TNFα (20 ng/ml); S, Smac mimetics (1 μM); Z, zVAD.fmk (1 μM). g Ripk1 +/+ and Ripk1 K376R/K376R immortalized MEFs that expressed with the Flag-tagged TAK1/TAB1 or TAK1-∆N were stimulated with TNFα (20 ng/ml) plus zVAD.fmk (10 μM) for different periods of time. The cell lysates were analyzed by western blotting with indicated antibodies. h Cell death of Ripk1 +/+ and Ripk1 K376R/K376R immortalized MEFs treated for 5 h with different stimulators was measured by SytoxGreen positivity. T: TNFα (20 ng/ml), Z: zVAD.fmk (10 μM), TAK1i: TAK1 inhibitor (500 μM), IKKi: IKK inhibitor(5 μM), MK2i: MK2 inhibitor (2 μM). In d , h , data are mean ± s.e.m. ( n = 3 independent cell samples for each genotype). Statistical significance was determined using a two-tailed unpaired t test, n.s., P > 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001
    Figure Legend Snippet: Ripk1 K376R/K376R mutation increases RIPK1 kinase activity. a Immortalized Ripk1 +/+ , Ripk1 K376R/K376R , and Ripk1 −/− MEFs were treated for the indicated time with TNFα (20 ng/ml). The K63-ubiquitylated proteins were isolated by K63-TUBEs and analyzed by western blot. b TNF-RSC was immunoprecipitated using anti-Flag resin in Ripk1 +/+ and Ripk1 K376R/K376R immortalized MEFs treated for the indicated time with Flag-TNFα (100 ng/ml). The immunocomplexes were analyzed by western blot with indicated antibodies. c Nuclear extracts were collected from Ripk1 +/+ , Ripk1 K376R/K376R , and Ripk1 −/− immortalized MEFs treated with TNFα (20 ng/ml) at indicated times and analyzed by western blotting with antibodies against p65 and PCNA. d Ripk1 +/+ and Ripk1 K376R/K376R immortalized MEFs that stably expressed with the Flag-tagged IκBα-SR were stimulated with TNFα (40 ng/ml) for different periods of time. Cell death was measured by SytoxGreen positivity. e , f Complex II was immunoprecipitated using RIPK1 antibody ( e ) or RIPK3 antibody ( f ) in Ripk1 +/+ , Ripk1 K376R/K376R , and Ripk1 −/− immortalized MEFs treated for the indicated time with TSZ ( e ) or TZ ( f ). The immunocomplexes were analyzed by western blotting with indicated antibodies. T, TNFα (20 ng/ml); S, Smac mimetics (1 μM); Z, zVAD.fmk (1 μM). g Ripk1 +/+ and Ripk1 K376R/K376R immortalized MEFs that expressed with the Flag-tagged TAK1/TAB1 or TAK1-∆N were stimulated with TNFα (20 ng/ml) plus zVAD.fmk (10 μM) for different periods of time. The cell lysates were analyzed by western blotting with indicated antibodies. h Cell death of Ripk1 +/+ and Ripk1 K376R/K376R immortalized MEFs treated for 5 h with different stimulators was measured by SytoxGreen positivity. T: TNFα (20 ng/ml), Z: zVAD.fmk (10 μM), TAK1i: TAK1 inhibitor (500 μM), IKKi: IKK inhibitor(5 μM), MK2i: MK2 inhibitor (2 μM). In d , h , data are mean ± s.e.m. ( n = 3 independent cell samples for each genotype). Statistical significance was determined using a two-tailed unpaired t test, n.s., P > 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001

    Techniques Used: Mutagenesis, Activity Assay, Isolation, Western Blot, Immunoprecipitation, Stable Transfection, Two Tailed Test


    Structured Review

    Tocris mk2 inhibitor
    Phosphorylated <t>MK2</t> (p-MK2) is overexpressed in head and neck tumor tissue compared to non-cancerous human tissue. A , Normal human head and neck tissue was obtained from the UNM Human Tissue Repository (HTR) via the IRB-approved study, INST 1310 (SRC 008-17). Standard immunohistochemistry was performed on 4-micron cut tissue sections staining for p-MK2. B , Primary head and neck cancer tissue obtained through institutional HTR using two different IRB approved protocols (University of New Mexico: HN009, HN009LN, HN011P, HN011T, HN013; University of Colorado Denver: CUHN002, CUHN004, CUHN0013, CUHN014) were cut and stained for p-MK2. Both p16 negative (p16−) and p16 positive (p16+) primary human HNSCC tissues were examined. Black bar denotes 50 μm size.
    Mk2 Inhibitor, supplied by Tocris, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mk2 inhibitor/product/Tocris
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mk2 inhibitor - by Bioz Stars, 2023-12
    94/100 stars

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    1) Product Images from "MAPKAPK2 (MK2) inhibition mediates radiation-induced inflammatory cytokine production and tumor growth in head and neck squamous cell carcinoma"

    Article Title: MAPKAPK2 (MK2) inhibition mediates radiation-induced inflammatory cytokine production and tumor growth in head and neck squamous cell carcinoma

    Journal: Oncogene

    doi: 10.1038/s41388-019-0945-9

    Phosphorylated MK2 (p-MK2) is overexpressed in head and neck tumor tissue compared to non-cancerous human tissue. A , Normal human head and neck tissue was obtained from the UNM Human Tissue Repository (HTR) via the IRB-approved study, INST 1310 (SRC 008-17). Standard immunohistochemistry was performed on 4-micron cut tissue sections staining for p-MK2. B , Primary head and neck cancer tissue obtained through institutional HTR using two different IRB approved protocols (University of New Mexico: HN009, HN009LN, HN011P, HN011T, HN013; University of Colorado Denver: CUHN002, CUHN004, CUHN0013, CUHN014) were cut and stained for p-MK2. Both p16 negative (p16−) and p16 positive (p16+) primary human HNSCC tissues were examined. Black bar denotes 50 μm size.
    Figure Legend Snippet: Phosphorylated MK2 (p-MK2) is overexpressed in head and neck tumor tissue compared to non-cancerous human tissue. A , Normal human head and neck tissue was obtained from the UNM Human Tissue Repository (HTR) via the IRB-approved study, INST 1310 (SRC 008-17). Standard immunohistochemistry was performed on 4-micron cut tissue sections staining for p-MK2. B , Primary head and neck cancer tissue obtained through institutional HTR using two different IRB approved protocols (University of New Mexico: HN009, HN009LN, HN011P, HN011T, HN013; University of Colorado Denver: CUHN002, CUHN004, CUHN0013, CUHN014) were cut and stained for p-MK2. Both p16 negative (p16−) and p16 positive (p16+) primary human HNSCC tissues were examined. Black bar denotes 50 μm size.

    Techniques Used: Immunohistochemistry, Staining

    High MK2 phosphorylation is a poor prognostic factor in HNSCC. A , Using an annotated oropharyngeal HNSCC tissue microarray, we queried whether HNSCC patients with Stage III, IV disease and high versus low p-MK2 levels impacted disease specific survival. Kaplan-Meier disease-specific survival curves were generated between p16+ [p-MK2 low (n = 33) versus MK2 high n = 29)] and p16− [p-MK2 low (n = 23) versus MK2 high (n = 26)] patients. Unlike the p16+ patients, the p16− patients demonstrated a significant survival difference between high versus low p-MK2 levels, p = 0.034. B , All tissue microarray slides were scanned at the highest digital resolution (40x) using the Aperio Imaging platform. Digitized slides were then imported into the HALO analysis system where a blinded pathologist and researcher helped to train the software program to differentiate between staining intensity (0+, 1+, 2+ and 3+) and tumor stroma versus the tumor itself. Representative p-MK2 immunohistochemistry and the respective HALO analyses was performed on p16− and p16+ samples. Representative samples here were further stratified between low versus high p-MK2 levels. Blue = 0+, yellow = 1+, orange = 1+; red = 3+. Slides were digitally rescaled from the 40x to 10x resolution. White bar denotes 100 μm size.
    Figure Legend Snippet: High MK2 phosphorylation is a poor prognostic factor in HNSCC. A , Using an annotated oropharyngeal HNSCC tissue microarray, we queried whether HNSCC patients with Stage III, IV disease and high versus low p-MK2 levels impacted disease specific survival. Kaplan-Meier disease-specific survival curves were generated between p16+ [p-MK2 low (n = 33) versus MK2 high n = 29)] and p16− [p-MK2 low (n = 23) versus MK2 high (n = 26)] patients. Unlike the p16+ patients, the p16− patients demonstrated a significant survival difference between high versus low p-MK2 levels, p = 0.034. B , All tissue microarray slides were scanned at the highest digital resolution (40x) using the Aperio Imaging platform. Digitized slides were then imported into the HALO analysis system where a blinded pathologist and researcher helped to train the software program to differentiate between staining intensity (0+, 1+, 2+ and 3+) and tumor stroma versus the tumor itself. Representative p-MK2 immunohistochemistry and the respective HALO analyses was performed on p16− and p16+ samples. Representative samples here were further stratified between low versus high p-MK2 levels. Blue = 0+, yellow = 1+, orange = 1+; red = 3+. Slides were digitally rescaled from the 40x to 10x resolution. White bar denotes 100 μm size.

    Techniques Used: Microarray, Generated, Imaging, Software, Staining, Immunohistochemistry

    RT leads to MK2 phosphorylation in mouse PDXs. A , p-MK2 immunohistochemistry staining of various F2 generation p16− and p16+ human PDX tumors, treated with or without RT (5Gy x 2). B , Quantitative HALO analyses for different representative PDX tumor samples looking at overall p-MK2 labeling between irradiated and non-irradiated samples demonstrated increased p-MK2 labeling compared to non-irradiated tissue.
    Figure Legend Snippet: RT leads to MK2 phosphorylation in mouse PDXs. A , p-MK2 immunohistochemistry staining of various F2 generation p16− and p16+ human PDX tumors, treated with or without RT (5Gy x 2). B , Quantitative HALO analyses for different representative PDX tumor samples looking at overall p-MK2 labeling between irradiated and non-irradiated samples demonstrated increased p-MK2 labeling compared to non-irradiated tissue.

    Techniques Used: Immunohistochemistry, Staining, Labeling, Irradiation

    Protein and mRNA profiles in HPV− and HPV+ HNSCC cell lines. A , p-MK2 protein expression in cell lines treated with RT, MK2i, or RT+MK2i. B , Inflammatory cytokine gene expression in cells treated with sham, RT, MK2i or RT+MK2i in multiple tumor cell lines. C , EMT gene expression in cells treated with sham, RT, MK2i or RT+MK2i in multiple tumor cell lines. Significance: * = p<0.05; ** = p<0.01; *** = p<0.001
    Figure Legend Snippet: Protein and mRNA profiles in HPV− and HPV+ HNSCC cell lines. A , p-MK2 protein expression in cell lines treated with RT, MK2i, or RT+MK2i. B , Inflammatory cytokine gene expression in cells treated with sham, RT, MK2i or RT+MK2i in multiple tumor cell lines. C , EMT gene expression in cells treated with sham, RT, MK2i or RT+MK2i in multiple tumor cell lines. Significance: * = p<0.05; ** = p<0.01; *** = p<0.001

    Techniques Used: Expressing

    MK2 siRNA blocks RT-induced MK2 and HSP27 phosphorylation in HPV− and HPV+ cell lines. HNSCC cell lines were treated with siRNA, with or without RT. A , Protein levels of MK2, p-MK2, HSP27, p-HSP27 and IL-6 and B , mRNA expression for MK2, IL-6, IL-1a, IL-1ß, TNF-a were examined. Significance: * = p<0.05; ** = p<0.01; *** = p<0.001
    Figure Legend Snippet: MK2 siRNA blocks RT-induced MK2 and HSP27 phosphorylation in HPV− and HPV+ cell lines. HNSCC cell lines were treated with siRNA, with or without RT. A , Protein levels of MK2, p-MK2, HSP27, p-HSP27 and IL-6 and B , mRNA expression for MK2, IL-6, IL-1a, IL-1ß, TNF-a were examined. Significance: * = p<0.05; ** = p<0.01; *** = p<0.001

    Techniques Used: Expressing

    MK2 pathway blockade enhances RT sensitivity and blocks RT-induced pro-tumorigenic cytokine production, in vivo . A and C , Fold change in HN009LN (p16+) and HN011T (p16−) tumor volumes in mice treated with excipient, RT, MK2i, or RT+MK2i. B and D , PDX tumor survival curves. Athymic nude mice xenografted with HN009LN ( A ) and HN011T ( C ) tumors were monitored and euthanized appropriately per IACUC approved protocol when their tumors reached 2000 mm 3 , tumors became ulcerated or animals became moribund. Kaplan-Meier survival curves were generated comparing excipient/control, MK2i, RT or RT+MK2i treatment arms. Survival was measured from the first day of treatment onwards. P values noted in the survival curve (n = 5-6 animals per arm). Significance: * = p<0.05; ** = p<0.01; *** = p<0.001.
    Figure Legend Snippet: MK2 pathway blockade enhances RT sensitivity and blocks RT-induced pro-tumorigenic cytokine production, in vivo . A and C , Fold change in HN009LN (p16+) and HN011T (p16−) tumor volumes in mice treated with excipient, RT, MK2i, or RT+MK2i. B and D , PDX tumor survival curves. Athymic nude mice xenografted with HN009LN ( A ) and HN011T ( C ) tumors were monitored and euthanized appropriately per IACUC approved protocol when their tumors reached 2000 mm 3 , tumors became ulcerated or animals became moribund. Kaplan-Meier survival curves were generated comparing excipient/control, MK2i, RT or RT+MK2i treatment arms. Survival was measured from the first day of treatment onwards. P values noted in the survival curve (n = 5-6 animals per arm). Significance: * = p<0.05; ** = p<0.01; *** = p<0.001.

    Techniques Used: In Vivo, Generated


    Structured Review

    Tocris mk2 inhibitor pf
    Mk2 Inhibitor Pf, supplied by Tocris, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mk2 inhibitor pf/product/Tocris
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mk2 inhibitor pf - by Bioz Stars, 2023-12
    94/100 stars

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    Structured Review

    Tocris protein kinase 2 mk2 inhibitor pf
    TGFβ induces post-translational modification of MRTF in a p38- and <t>MK2-dependent</t> manner. A and B, C3H/10T1/2 cells were pretreated with vehicle (DMSO) or DORA (A) or AKTi (B) and, where indicated, exposed to TGFβ for 6 h. Whole-cell extracts were run in 6% SDS gels, and the relative shift is the molecular mass of MRTF (see dotted lines) was determined and normalized to the change detected in the vehicle-treated controls as described under “Experimental procedures.” C and D, cells were transfected with 300 nm non-related (siNR) or α (100 nm)-, β (100 nm)-, and γ (100 nm)-p38 siRNA (sip38) for 48 h and then, where indicated, exposed to TGFβ for 6 h. Cortactin was used as loading control. TAZ expression (C) and the relative molecular shift of MRTF (D) were assessed by Western blotting (n = 3) as in Figs. 1A and ​and88B, respectively. α-Actinin (α-act) was used as loading control. E, cells were cotransfected with 3DA firefly reporter and Renilla luciferase and, where indicated (panel iii) with Smad3 siRNA or NR siRNA for 24 h. Subsequently cells were preincubated with vehicle (DMSO) or DORA (panel i) or AKTi (panel ii) for 30 min. Cells were then exposed to TGFβ for 6 h, as indicated. Normalized luciferase activities were determined (n = 3 for each condition). F and G, C3H/10T1/2 cells were pretreated with vehicle or the MK2 inhibitor <t>PF-3644022</t> (PF, 10 μm) followed by 6 h of treatment with TGFβ as indicated and then processed for Western blotting to assess TAZ expression (F) or the shift of MRTF (G) (n = 3). H and I, conditions were as in F and G, except the cells were pretreated where indicated with the antifibrotic drug, pirfenidone (Pirf, 1 mg/ml). n.s., non-significant.
    Protein Kinase 2 Mk2 Inhibitor Pf, supplied by Tocris, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/protein kinase 2 mk2 inhibitor pf/product/Tocris
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    protein kinase 2 mk2 inhibitor pf - by Bioz Stars, 2023-12
    94/100 stars

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    1) Product Images from "TGF-β1 regulates the expression and transcriptional activity of TAZ protein via a Smad3-independent, myocardin-related transcription factor-mediated mechanism"

    Article Title: TGF-β1 regulates the expression and transcriptional activity of TAZ protein via a Smad3-independent, myocardin-related transcription factor-mediated mechanism

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M117.780502

    TGFβ induces post-translational modification of MRTF in a p38- and MK2-dependent manner. A and B, C3H/10T1/2 cells were pretreated with vehicle (DMSO) or DORA (A) or AKTi (B) and, where indicated, exposed to TGFβ for 6 h. Whole-cell extracts were run in 6% SDS gels, and the relative shift is the molecular mass of MRTF (see dotted lines) was determined and normalized to the change detected in the vehicle-treated controls as described under “Experimental procedures.” C and D, cells were transfected with 300 nm non-related (siNR) or α (100 nm)-, β (100 nm)-, and γ (100 nm)-p38 siRNA (sip38) for 48 h and then, where indicated, exposed to TGFβ for 6 h. Cortactin was used as loading control. TAZ expression (C) and the relative molecular shift of MRTF (D) were assessed by Western blotting (n = 3) as in Figs. 1A and ​and88B, respectively. α-Actinin (α-act) was used as loading control. E, cells were cotransfected with 3DA firefly reporter and Renilla luciferase and, where indicated (panel iii) with Smad3 siRNA or NR siRNA for 24 h. Subsequently cells were preincubated with vehicle (DMSO) or DORA (panel i) or AKTi (panel ii) for 30 min. Cells were then exposed to TGFβ for 6 h, as indicated. Normalized luciferase activities were determined (n = 3 for each condition). F and G, C3H/10T1/2 cells were pretreated with vehicle or the MK2 inhibitor PF-3644022 (PF, 10 μm) followed by 6 h of treatment with TGFβ as indicated and then processed for Western blotting to assess TAZ expression (F) or the shift of MRTF (G) (n = 3). H and I, conditions were as in F and G, except the cells were pretreated where indicated with the antifibrotic drug, pirfenidone (Pirf, 1 mg/ml). n.s., non-significant.
    Figure Legend Snippet: TGFβ induces post-translational modification of MRTF in a p38- and MK2-dependent manner. A and B, C3H/10T1/2 cells were pretreated with vehicle (DMSO) or DORA (A) or AKTi (B) and, where indicated, exposed to TGFβ for 6 h. Whole-cell extracts were run in 6% SDS gels, and the relative shift is the molecular mass of MRTF (see dotted lines) was determined and normalized to the change detected in the vehicle-treated controls as described under “Experimental procedures.” C and D, cells were transfected with 300 nm non-related (siNR) or α (100 nm)-, β (100 nm)-, and γ (100 nm)-p38 siRNA (sip38) for 48 h and then, where indicated, exposed to TGFβ for 6 h. Cortactin was used as loading control. TAZ expression (C) and the relative molecular shift of MRTF (D) were assessed by Western blotting (n = 3) as in Figs. 1A and ​and88B, respectively. α-Actinin (α-act) was used as loading control. E, cells were cotransfected with 3DA firefly reporter and Renilla luciferase and, where indicated (panel iii) with Smad3 siRNA or NR siRNA for 24 h. Subsequently cells were preincubated with vehicle (DMSO) or DORA (panel i) or AKTi (panel ii) for 30 min. Cells were then exposed to TGFβ for 6 h, as indicated. Normalized luciferase activities were determined (n = 3 for each condition). F and G, C3H/10T1/2 cells were pretreated with vehicle or the MK2 inhibitor PF-3644022 (PF, 10 μm) followed by 6 h of treatment with TGFβ as indicated and then processed for Western blotting to assess TAZ expression (F) or the shift of MRTF (G) (n = 3). H and I, conditions were as in F and G, except the cells were pretreated where indicated with the antifibrotic drug, pirfenidone (Pirf, 1 mg/ml). n.s., non-significant.

    Techniques Used: Modification, Transfection, Expressing, Western Blot, Luciferase

    protein kinase 2 mk2 inhibitor pf 3644022  (Tocris)

     
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    Tocris protein kinase 2 mk2 inhibitor pf 3644022
    Protein Kinase 2 Mk2 Inhibitor Pf 3644022, supplied by Tocris, 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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    Average 86 stars, based on 1 article reviews
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    Tocris mk2 inhibitor pf 3644022
    Mk2 Inhibitor Pf 3644022, supplied by Tocris, 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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    Tocris atp competitive mapkapk2 mk2 inhibitor pf 3644022
    Atp Competitive Mapkapk2 Mk2 Inhibitor Pf 3644022, supplied by Tocris, 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/atp competitive mapkapk2 mk2 inhibitor pf 3644022/product/Tocris
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    Tocris mk2 inhibitor pf
    Mk2 Inhibitor Pf, supplied by Tocris, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mk2 inhibitor pf/product/Tocris
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    Ripk1 K376R/K376R mutation increases RIPK1 kinase activity. a Immortalized Ripk1 +/+ , Ripk1 K376R/K376R , and Ripk1 −/− MEFs were treated for the indicated time with TNFα (20 ng/ml). The K63-ubiquitylated proteins were isolated by K63-TUBEs and analyzed by western blot. b TNF-RSC was immunoprecipitated using anti-Flag resin in Ripk1 +/+ and Ripk1 K376R/K376R immortalized MEFs treated for the indicated time with Flag-TNFα (100 ng/ml). The immunocomplexes were analyzed by western blot with indicated antibodies. c Nuclear extracts were collected from Ripk1 +/+ , Ripk1 K376R/K376R , and Ripk1 −/− immortalized MEFs treated with TNFα (20 ng/ml) at indicated times and analyzed by western blotting with antibodies against p65 and PCNA. d Ripk1 +/+ and Ripk1 K376R/K376R immortalized MEFs that stably expressed with the Flag-tagged IκBα-SR were stimulated with TNFα (40 ng/ml) for different periods of time. Cell death was measured by SytoxGreen positivity. e , f Complex II was immunoprecipitated using RIPK1 antibody ( e ) or RIPK3 antibody ( f ) in Ripk1 +/+ , Ripk1 K376R/K376R , and Ripk1 −/− immortalized MEFs treated for the indicated time with TSZ ( e ) or TZ ( f ). The immunocomplexes were analyzed by western blotting with indicated antibodies. T, TNFα (20 ng/ml); S, Smac mimetics (1 μM); Z, zVAD.fmk (1 μM). g Ripk1 +/+ and Ripk1 K376R/K376R immortalized MEFs that expressed with the Flag-tagged TAK1/TAB1 or TAK1-∆N were stimulated with TNFα (20 ng/ml) plus zVAD.fmk (10 μM) for different periods of time. The cell lysates were analyzed by western blotting with indicated antibodies. h Cell death of Ripk1 +/+ and Ripk1 K376R/K376R immortalized MEFs treated for 5 h with different stimulators was measured by SytoxGreen positivity. T: TNFα (20 ng/ml), Z: zVAD.fmk (10 μM), TAK1i: TAK1 inhibitor (500 μM), IKKi: IKK inhibitor(5 μM), MK2i: <t>MK2</t> inhibitor (2 μM). In d , h , data are mean ± s.e.m. ( n = 3 independent cell samples for each genotype). Statistical significance was determined using a two-tailed unpaired t test, n.s., P > 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001
    Mk2 Inhibitor, supplied by Tocris, 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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    TGFβ induces post-translational modification of MRTF in a p38- and <t>MK2-dependent</t> manner. A and B, C3H/10T1/2 cells were pretreated with vehicle (DMSO) or DORA (A) or AKTi (B) and, where indicated, exposed to TGFβ for 6 h. Whole-cell extracts were run in 6% SDS gels, and the relative shift is the molecular mass of MRTF (see dotted lines) was determined and normalized to the change detected in the vehicle-treated controls as described under “Experimental procedures.” C and D, cells were transfected with 300 nm non-related (siNR) or α (100 nm)-, β (100 nm)-, and γ (100 nm)-p38 siRNA (sip38) for 48 h and then, where indicated, exposed to TGFβ for 6 h. Cortactin was used as loading control. TAZ expression (C) and the relative molecular shift of MRTF (D) were assessed by Western blotting (n = 3) as in Figs. 1A and ​and88B, respectively. α-Actinin (α-act) was used as loading control. E, cells were cotransfected with 3DA firefly reporter and Renilla luciferase and, where indicated (panel iii) with Smad3 siRNA or NR siRNA for 24 h. Subsequently cells were preincubated with vehicle (DMSO) or DORA (panel i) or AKTi (panel ii) for 30 min. Cells were then exposed to TGFβ for 6 h, as indicated. Normalized luciferase activities were determined (n = 3 for each condition). F and G, C3H/10T1/2 cells were pretreated with vehicle or the MK2 inhibitor <t>PF-3644022</t> (PF, 10 μm) followed by 6 h of treatment with TGFβ as indicated and then processed for Western blotting to assess TAZ expression (F) or the shift of MRTF (G) (n = 3). H and I, conditions were as in F and G, except the cells were pretreated where indicated with the antifibrotic drug, pirfenidone (Pirf, 1 mg/ml). n.s., non-significant.
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    TGFβ induces post-translational modification of MRTF in a p38- and <t>MK2-dependent</t> manner. A and B, C3H/10T1/2 cells were pretreated with vehicle (DMSO) or DORA (A) or AKTi (B) and, where indicated, exposed to TGFβ for 6 h. Whole-cell extracts were run in 6% SDS gels, and the relative shift is the molecular mass of MRTF (see dotted lines) was determined and normalized to the change detected in the vehicle-treated controls as described under “Experimental procedures.” C and D, cells were transfected with 300 nm non-related (siNR) or α (100 nm)-, β (100 nm)-, and γ (100 nm)-p38 siRNA (sip38) for 48 h and then, where indicated, exposed to TGFβ for 6 h. Cortactin was used as loading control. TAZ expression (C) and the relative molecular shift of MRTF (D) were assessed by Western blotting (n = 3) as in Figs. 1A and ​and88B, respectively. α-Actinin (α-act) was used as loading control. E, cells were cotransfected with 3DA firefly reporter and Renilla luciferase and, where indicated (panel iii) with Smad3 siRNA or NR siRNA for 24 h. Subsequently cells were preincubated with vehicle (DMSO) or DORA (panel i) or AKTi (panel ii) for 30 min. Cells were then exposed to TGFβ for 6 h, as indicated. Normalized luciferase activities were determined (n = 3 for each condition). F and G, C3H/10T1/2 cells were pretreated with vehicle or the MK2 inhibitor <t>PF-3644022</t> (PF, 10 μm) followed by 6 h of treatment with TGFβ as indicated and then processed for Western blotting to assess TAZ expression (F) or the shift of MRTF (G) (n = 3). H and I, conditions were as in F and G, except the cells were pretreated where indicated with the antifibrotic drug, pirfenidone (Pirf, 1 mg/ml). n.s., non-significant.
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    Image Search Results


    Ripk1 K376R/K376R mutation increases RIPK1 kinase activity. a Immortalized Ripk1 +/+ , Ripk1 K376R/K376R , and Ripk1 −/− MEFs were treated for the indicated time with TNFα (20 ng/ml). The K63-ubiquitylated proteins were isolated by K63-TUBEs and analyzed by western blot. b TNF-RSC was immunoprecipitated using anti-Flag resin in Ripk1 +/+ and Ripk1 K376R/K376R immortalized MEFs treated for the indicated time with Flag-TNFα (100 ng/ml). The immunocomplexes were analyzed by western blot with indicated antibodies. c Nuclear extracts were collected from Ripk1 +/+ , Ripk1 K376R/K376R , and Ripk1 −/− immortalized MEFs treated with TNFα (20 ng/ml) at indicated times and analyzed by western blotting with antibodies against p65 and PCNA. d Ripk1 +/+ and Ripk1 K376R/K376R immortalized MEFs that stably expressed with the Flag-tagged IκBα-SR were stimulated with TNFα (40 ng/ml) for different periods of time. Cell death was measured by SytoxGreen positivity. e , f Complex II was immunoprecipitated using RIPK1 antibody ( e ) or RIPK3 antibody ( f ) in Ripk1 +/+ , Ripk1 K376R/K376R , and Ripk1 −/− immortalized MEFs treated for the indicated time with TSZ ( e ) or TZ ( f ). The immunocomplexes were analyzed by western blotting with indicated antibodies. T, TNFα (20 ng/ml); S, Smac mimetics (1 μM); Z, zVAD.fmk (1 μM). g Ripk1 +/+ and Ripk1 K376R/K376R immortalized MEFs that expressed with the Flag-tagged TAK1/TAB1 or TAK1-∆N were stimulated with TNFα (20 ng/ml) plus zVAD.fmk (10 μM) for different periods of time. The cell lysates were analyzed by western blotting with indicated antibodies. h Cell death of Ripk1 +/+ and Ripk1 K376R/K376R immortalized MEFs treated for 5 h with different stimulators was measured by SytoxGreen positivity. T: TNFα (20 ng/ml), Z: zVAD.fmk (10 μM), TAK1i: TAK1 inhibitor (500 μM), IKKi: IKK inhibitor(5 μM), MK2i: MK2 inhibitor (2 μM). In d , h , data are mean ± s.e.m. ( n = 3 independent cell samples for each genotype). Statistical significance was determined using a two-tailed unpaired t test, n.s., P > 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001

    Journal: Nature Communications

    Article Title: K63-linked ubiquitination regulates RIPK1 kinase activity to prevent cell death during embryogenesis and inflammation

    doi: 10.1038/s41467-019-12033-8

    Figure Lengend Snippet: Ripk1 K376R/K376R mutation increases RIPK1 kinase activity. a Immortalized Ripk1 +/+ , Ripk1 K376R/K376R , and Ripk1 −/− MEFs were treated for the indicated time with TNFα (20 ng/ml). The K63-ubiquitylated proteins were isolated by K63-TUBEs and analyzed by western blot. b TNF-RSC was immunoprecipitated using anti-Flag resin in Ripk1 +/+ and Ripk1 K376R/K376R immortalized MEFs treated for the indicated time with Flag-TNFα (100 ng/ml). The immunocomplexes were analyzed by western blot with indicated antibodies. c Nuclear extracts were collected from Ripk1 +/+ , Ripk1 K376R/K376R , and Ripk1 −/− immortalized MEFs treated with TNFα (20 ng/ml) at indicated times and analyzed by western blotting with antibodies against p65 and PCNA. d Ripk1 +/+ and Ripk1 K376R/K376R immortalized MEFs that stably expressed with the Flag-tagged IκBα-SR were stimulated with TNFα (40 ng/ml) for different periods of time. Cell death was measured by SytoxGreen positivity. e , f Complex II was immunoprecipitated using RIPK1 antibody ( e ) or RIPK3 antibody ( f ) in Ripk1 +/+ , Ripk1 K376R/K376R , and Ripk1 −/− immortalized MEFs treated for the indicated time with TSZ ( e ) or TZ ( f ). The immunocomplexes were analyzed by western blotting with indicated antibodies. T, TNFα (20 ng/ml); S, Smac mimetics (1 μM); Z, zVAD.fmk (1 μM). g Ripk1 +/+ and Ripk1 K376R/K376R immortalized MEFs that expressed with the Flag-tagged TAK1/TAB1 or TAK1-∆N were stimulated with TNFα (20 ng/ml) plus zVAD.fmk (10 μM) for different periods of time. The cell lysates were analyzed by western blotting with indicated antibodies. h Cell death of Ripk1 +/+ and Ripk1 K376R/K376R immortalized MEFs treated for 5 h with different stimulators was measured by SytoxGreen positivity. T: TNFα (20 ng/ml), Z: zVAD.fmk (10 μM), TAK1i: TAK1 inhibitor (500 μM), IKKi: IKK inhibitor(5 μM), MK2i: MK2 inhibitor (2 μM). In d , h , data are mean ± s.e.m. ( n = 3 independent cell samples for each genotype). Statistical significance was determined using a two-tailed unpaired t test, n.s., P > 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001

    Article Snippet: MK2 inhibitor (4279/10) was obtained from Tocris Bioscience. zVAD.fmk (C1202) was obtained from Beyotime.

    Techniques: Mutagenesis, Activity Assay, Isolation, Western Blot, Immunoprecipitation, Stable Transfection, Two Tailed Test

    TGFβ induces post-translational modification of MRTF in a p38- and MK2-dependent manner. A and B, C3H/10T1/2 cells were pretreated with vehicle (DMSO) or DORA (A) or AKTi (B) and, where indicated, exposed to TGFβ for 6 h. Whole-cell extracts were run in 6% SDS gels, and the relative shift is the molecular mass of MRTF (see dotted lines) was determined and normalized to the change detected in the vehicle-treated controls as described under “Experimental procedures.” C and D, cells were transfected with 300 nm non-related (siNR) or α (100 nm)-, β (100 nm)-, and γ (100 nm)-p38 siRNA (sip38) for 48 h and then, where indicated, exposed to TGFβ for 6 h. Cortactin was used as loading control. TAZ expression (C) and the relative molecular shift of MRTF (D) were assessed by Western blotting (n = 3) as in Figs. 1A and ​and88B, respectively. α-Actinin (α-act) was used as loading control. E, cells were cotransfected with 3DA firefly reporter and Renilla luciferase and, where indicated (panel iii) with Smad3 siRNA or NR siRNA for 24 h. Subsequently cells were preincubated with vehicle (DMSO) or DORA (panel i) or AKTi (panel ii) for 30 min. Cells were then exposed to TGFβ for 6 h, as indicated. Normalized luciferase activities were determined (n = 3 for each condition). F and G, C3H/10T1/2 cells were pretreated with vehicle or the MK2 inhibitor PF-3644022 (PF, 10 μm) followed by 6 h of treatment with TGFβ as indicated and then processed for Western blotting to assess TAZ expression (F) or the shift of MRTF (G) (n = 3). H and I, conditions were as in F and G, except the cells were pretreated where indicated with the antifibrotic drug, pirfenidone (Pirf, 1 mg/ml). n.s., non-significant.

    Journal: The Journal of Biological Chemistry

    Article Title: TGF-β1 regulates the expression and transcriptional activity of TAZ protein via a Smad3-independent, myocardin-related transcription factor-mediated mechanism

    doi: 10.1074/jbc.M117.780502

    Figure Lengend Snippet: TGFβ induces post-translational modification of MRTF in a p38- and MK2-dependent manner. A and B, C3H/10T1/2 cells were pretreated with vehicle (DMSO) or DORA (A) or AKTi (B) and, where indicated, exposed to TGFβ for 6 h. Whole-cell extracts were run in 6% SDS gels, and the relative shift is the molecular mass of MRTF (see dotted lines) was determined and normalized to the change detected in the vehicle-treated controls as described under “Experimental procedures.” C and D, cells were transfected with 300 nm non-related (siNR) or α (100 nm)-, β (100 nm)-, and γ (100 nm)-p38 siRNA (sip38) for 48 h and then, where indicated, exposed to TGFβ for 6 h. Cortactin was used as loading control. TAZ expression (C) and the relative molecular shift of MRTF (D) were assessed by Western blotting (n = 3) as in Figs. 1A and ​and88B, respectively. α-Actinin (α-act) was used as loading control. E, cells were cotransfected with 3DA firefly reporter and Renilla luciferase and, where indicated (panel iii) with Smad3 siRNA or NR siRNA for 24 h. Subsequently cells were preincubated with vehicle (DMSO) or DORA (panel i) or AKTi (panel ii) for 30 min. Cells were then exposed to TGFβ for 6 h, as indicated. Normalized luciferase activities were determined (n = 3 for each condition). F and G, C3H/10T1/2 cells were pretreated with vehicle or the MK2 inhibitor PF-3644022 (PF, 10 μm) followed by 6 h of treatment with TGFβ as indicated and then processed for Western blotting to assess TAZ expression (F) or the shift of MRTF (G) (n = 3). H and I, conditions were as in F and G, except the cells were pretreated where indicated with the antifibrotic drug, pirfenidone (Pirf, 1 mg/ml). n.s., non-significant.

    Article Snippet: The p38 MAPK inhibitor doramapimod (BIRB-76) was from Cayman Chemical Co. (Ann Arbor, MI); MEK1/2 inhibitor U0126 was from Alexis Biochemicals (San Diego, CA); Smad3 inhibitor SIS3 and NADPH oxidase inhibitor VAS2870 were from Calbiochem/EDM Millipore (Billerica, MA); mTORC1 inhibitor rapamycin was from Abcam (Cambridge, UK); Food and Drug Administration-approved anti-fibrotic drug pirfenidone and MAPK-activated protein kinase-2 (MK2) inhibitor PF-3644022 were from Tocris (Minneapolis, MN).

    Techniques: Modification, Transfection, Expressing, Western Blot, Luciferase