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    • mk2 inhibitor  (Tocris)
    94
    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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    pf 3644022  (Millipore)
    86
    Millipore pf 3644022
    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
    Pf 3644022, supplied by Millipore, 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/pf 3644022/product/Millipore
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pf 3644022 - by Bioz Stars, 2023-12
    86/100 stars
    		  Buy from Supplier
    pf 3644022  (Pfizer Inc)
    86
    Pfizer Inc pf 3644022
    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
    Pf 3644022, supplied by Pfizer 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/pf 3644022/product/Pfizer Inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pf 3644022 - by Bioz Stars, 2023-12
    86/100 stars
    		  Buy from Supplier
    mk2 inhibitor pf 3644022  (R&D Systems)
    86
    R&D Systems mk2 inhibitor pf 3644022
    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 Pf 3644022, supplied by R&D Systems, 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/mk2 inhibitor pf 3644022/product/R&D Systems
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mk2 inhibitor pf 3644022 - by Bioz Stars, 2023-12
    86/100 stars
    		  Buy from Supplier

    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