6 thio gtp  (Jena Bioscience)


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  • 91
    Name:
    6 Thio GTP
    Description:

    Catalog Number:
    nu-1106l
    Price:
    352.15
    Applications:
    Allosteric effect on E.coli CTP-synthase[1] Inhibition of Vac-1-Rac signalling[2] Immunosuppression by blockade of GTPase activation[3, 4] Immunosuppressive drug (transplantation, inflammation)[1, 4]
    Purity:
    ≥ 95 % (HPLC)
    Category:
    Nucleotides Nucleosides
    Buy from Supplier


    Structured Review

    Jena Bioscience 6 thio gtp
    ( a ) Rac1 activation assay in activated primary T cells. Purified CD4 + T lymphocytes were stimulated with IL-2 and antibodies to CD3 or IL-2 plus antibodies to CD3 and CD28 for 3 days. <t>GTP-bound</t> Rac1 (Rac1-GTP) was analyzed using PAK to determine Rac1 activation. CD28 costimulation led to induction of Rac1 activation in primary T cells. ( b ) Azathioprine and 6-MP suppress Rac1 activation. Purified CD4 + T lymphocytes were stimulated in the presence or absence of azathioprine or 6-MP for 3 days, as indicated. GTP-bound Rac1 was analyzed using PAK to determine Rac1 activation. Azathioprine treatment led to a reduction of CD28-dependent Rac1 activation. One representative experiment out of five is shown. ( c ) 6-MP and 6-TG fail to modulate Ras activation. GTP-bound Ras (Ras-GTP) was analyzed using Raf RGD to determine Ras activation. Azathioprine and its metabolites did not affect Ras activation. One representative experiment of three is shown. ( d ) Downregulation of STAT-3 in primary T cells upon treatment with azathioprine or 6-MP. Purified CD4 + ), GTP-bound Rac1 was obtained using PAK, and STAT-3 levels were determined by immunoblotting. ( e ) Competition of GTP binding to Rac1 or Ras by <t>6-Thio-GTP.</t> Recombinant Rac1 or Ras was incubated with radiolabeled GTP ([ 3 H]GTP) and increasing amounts of chemically synthesized 6-Thio-GTP (0–500 μM). Next, Rac1 was obtained using PAK-1 agarose, followed by analysis of [ 3 H]GTP-bound Rac1 by scintillation counting. Similarly, Ras was obtained using Raf RGD agarose followed by analysis of [ 3 H]GTP-bound Ras by scintillation counting. 6-Thio-GTP led to a concentration-dependent suppression of [ 3 H]GTP-bound Rac1 but had little effect on [ 3 H]GTP-bound Ras.

    https://www.bioz.com/result/6 thio gtp/product/Jena Bioscience
    Average 91 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    6 thio gtp - by Bioz Stars, 2020-08
    91/100 stars

    Images

    1) Product Images from "CD28-dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes"

    Article Title: CD28-dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes

    Journal: Journal of Clinical Investigation

    doi: 10.1172/JCI200316432

    ( a ) Rac1 activation assay in activated primary T cells. Purified CD4 + T lymphocytes were stimulated with IL-2 and antibodies to CD3 or IL-2 plus antibodies to CD3 and CD28 for 3 days. GTP-bound Rac1 (Rac1-GTP) was analyzed using PAK to determine Rac1 activation. CD28 costimulation led to induction of Rac1 activation in primary T cells. ( b ) Azathioprine and 6-MP suppress Rac1 activation. Purified CD4 + T lymphocytes were stimulated in the presence or absence of azathioprine or 6-MP for 3 days, as indicated. GTP-bound Rac1 was analyzed using PAK to determine Rac1 activation. Azathioprine treatment led to a reduction of CD28-dependent Rac1 activation. One representative experiment out of five is shown. ( c ) 6-MP and 6-TG fail to modulate Ras activation. GTP-bound Ras (Ras-GTP) was analyzed using Raf RGD to determine Ras activation. Azathioprine and its metabolites did not affect Ras activation. One representative experiment of three is shown. ( d ) Downregulation of STAT-3 in primary T cells upon treatment with azathioprine or 6-MP. Purified CD4 + ), GTP-bound Rac1 was obtained using PAK, and STAT-3 levels were determined by immunoblotting. ( e ) Competition of GTP binding to Rac1 or Ras by 6-Thio-GTP. Recombinant Rac1 or Ras was incubated with radiolabeled GTP ([ 3 H]GTP) and increasing amounts of chemically synthesized 6-Thio-GTP (0–500 μM). Next, Rac1 was obtained using PAK-1 agarose, followed by analysis of [ 3 H]GTP-bound Rac1 by scintillation counting. Similarly, Ras was obtained using Raf RGD agarose followed by analysis of [ 3 H]GTP-bound Ras by scintillation counting. 6-Thio-GTP led to a concentration-dependent suppression of [ 3 H]GTP-bound Rac1 but had little effect on [ 3 H]GTP-bound Ras.
    Figure Legend Snippet: ( a ) Rac1 activation assay in activated primary T cells. Purified CD4 + T lymphocytes were stimulated with IL-2 and antibodies to CD3 or IL-2 plus antibodies to CD3 and CD28 for 3 days. GTP-bound Rac1 (Rac1-GTP) was analyzed using PAK to determine Rac1 activation. CD28 costimulation led to induction of Rac1 activation in primary T cells. ( b ) Azathioprine and 6-MP suppress Rac1 activation. Purified CD4 + T lymphocytes were stimulated in the presence or absence of azathioprine or 6-MP for 3 days, as indicated. GTP-bound Rac1 was analyzed using PAK to determine Rac1 activation. Azathioprine treatment led to a reduction of CD28-dependent Rac1 activation. One representative experiment out of five is shown. ( c ) 6-MP and 6-TG fail to modulate Ras activation. GTP-bound Ras (Ras-GTP) was analyzed using Raf RGD to determine Ras activation. Azathioprine and its metabolites did not affect Ras activation. One representative experiment of three is shown. ( d ) Downregulation of STAT-3 in primary T cells upon treatment with azathioprine or 6-MP. Purified CD4 + ), GTP-bound Rac1 was obtained using PAK, and STAT-3 levels were determined by immunoblotting. ( e ) Competition of GTP binding to Rac1 or Ras by 6-Thio-GTP. Recombinant Rac1 or Ras was incubated with radiolabeled GTP ([ 3 H]GTP) and increasing amounts of chemically synthesized 6-Thio-GTP (0–500 μM). Next, Rac1 was obtained using PAK-1 agarose, followed by analysis of [ 3 H]GTP-bound Rac1 by scintillation counting. Similarly, Ras was obtained using Raf RGD agarose followed by analysis of [ 3 H]GTP-bound Ras by scintillation counting. 6-Thio-GTP led to a concentration-dependent suppression of [ 3 H]GTP-bound Rac1 but had little effect on [ 3 H]GTP-bound Ras.

    Techniques Used: Activation Assay, Purification, Binding Assay, Recombinant, Incubation, Synthesized, Concentration Assay

    2) Product Images from "CD28-dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes"

    Article Title: CD28-dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes

    Journal: Journal of Clinical Investigation

    doi: 10.1172/JCI200316432

    ( a ) Rac1 activation assay in activated primary T cells. Purified CD4 + T lymphocytes were stimulated with IL-2 and antibodies to CD3 or IL-2 plus antibodies to CD3 and CD28 for 3 days. GTP-bound Rac1 (Rac1-GTP) was analyzed using PAK to determine Rac1 activation. CD28 costimulation led to induction of Rac1 activation in primary T cells. ( b ) Azathioprine and 6-MP suppress Rac1 activation. Purified CD4 + T lymphocytes were stimulated in the presence or absence of azathioprine or 6-MP for 3 days, as indicated. GTP-bound Rac1 was analyzed using PAK to determine Rac1 activation. Azathioprine treatment led to a reduction of CD28-dependent Rac1 activation. One representative experiment out of five is shown. ( c ) 6-MP and 6-TG fail to modulate Ras activation. GTP-bound Ras (Ras-GTP) was analyzed using Raf RGD to determine Ras activation. Azathioprine and its metabolites did not affect Ras activation. One representative experiment of three is shown. ( d ) Downregulation of STAT-3 in primary T cells upon treatment with azathioprine or 6-MP. Purified CD4 + ), GTP-bound Rac1 was obtained using PAK, and STAT-3 levels were determined by immunoblotting. ( e ) Competition of GTP binding to Rac1 or Ras by 6-Thio-GTP. Recombinant Rac1 or Ras was incubated with radiolabeled GTP ([ 3 H]GTP) and increasing amounts of chemically synthesized 6-Thio-GTP (0–500 μM). Next, Rac1 was obtained using PAK-1 agarose, followed by analysis of [ 3 H]GTP-bound Rac1 by scintillation counting. Similarly, Ras was obtained using Raf RGD agarose followed by analysis of [ 3 H]GTP-bound Ras by scintillation counting. 6-Thio-GTP led to a concentration-dependent suppression of [ 3 H]GTP-bound Rac1 but had little effect on [ 3 H]GTP-bound Ras.
    Figure Legend Snippet: ( a ) Rac1 activation assay in activated primary T cells. Purified CD4 + T lymphocytes were stimulated with IL-2 and antibodies to CD3 or IL-2 plus antibodies to CD3 and CD28 for 3 days. GTP-bound Rac1 (Rac1-GTP) was analyzed using PAK to determine Rac1 activation. CD28 costimulation led to induction of Rac1 activation in primary T cells. ( b ) Azathioprine and 6-MP suppress Rac1 activation. Purified CD4 + T lymphocytes were stimulated in the presence or absence of azathioprine or 6-MP for 3 days, as indicated. GTP-bound Rac1 was analyzed using PAK to determine Rac1 activation. Azathioprine treatment led to a reduction of CD28-dependent Rac1 activation. One representative experiment out of five is shown. ( c ) 6-MP and 6-TG fail to modulate Ras activation. GTP-bound Ras (Ras-GTP) was analyzed using Raf RGD to determine Ras activation. Azathioprine and its metabolites did not affect Ras activation. One representative experiment of three is shown. ( d ) Downregulation of STAT-3 in primary T cells upon treatment with azathioprine or 6-MP. Purified CD4 + ), GTP-bound Rac1 was obtained using PAK, and STAT-3 levels were determined by immunoblotting. ( e ) Competition of GTP binding to Rac1 or Ras by 6-Thio-GTP. Recombinant Rac1 or Ras was incubated with radiolabeled GTP ([ 3 H]GTP) and increasing amounts of chemically synthesized 6-Thio-GTP (0–500 μM). Next, Rac1 was obtained using PAK-1 agarose, followed by analysis of [ 3 H]GTP-bound Rac1 by scintillation counting. Similarly, Ras was obtained using Raf RGD agarose followed by analysis of [ 3 H]GTP-bound Ras by scintillation counting. 6-Thio-GTP led to a concentration-dependent suppression of [ 3 H]GTP-bound Rac1 but had little effect on [ 3 H]GTP-bound Ras.

    Techniques Used: Activation Assay, Purification, Binding Assay, Recombinant, Incubation, Synthesized, Concentration Assay

    3) Product Images from "T cell receptor-driven transendothelial migration of human effector memory CD4 T cells involves Vav, Rac and Myosin IIA"

    Article Title: T cell receptor-driven transendothelial migration of human effector memory CD4 T cells involves Vav, Rac and Myosin IIA

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1201817

    Vav is required for TCR-driven TEM. A. TEM assays of EM CD4 T cells treated with vehicle and 5 μM 6-Thio GTP for 44 hours. Graphs show %TEM of cells lacking the Vβ2TCR (VB2−, which do not interact with TSST-1 and transmigrate in
    Figure Legend Snippet: Vav is required for TCR-driven TEM. A. TEM assays of EM CD4 T cells treated with vehicle and 5 μM 6-Thio GTP for 44 hours. Graphs show %TEM of cells lacking the Vβ2TCR (VB2−, which do not interact with TSST-1 and transmigrate in

    Techniques Used: Transmission Electron Microscopy

    4) Product Images from "CD28-dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes"

    Article Title: CD28-dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes

    Journal: Journal of Clinical Investigation

    doi: 10.1172/JCI200316432

    ( a ) Rac1 activation assay in activated primary T cells. Purified CD4 + T lymphocytes were stimulated with IL-2 and antibodies to CD3 or IL-2 plus antibodies to CD3 and CD28 for 3 days. GTP-bound Rac1 (Rac1-GTP) was analyzed using PAK to determine Rac1 activation. CD28 costimulation led to induction of Rac1 activation in primary T cells. ( b ) Azathioprine and 6-MP suppress Rac1 activation. Purified CD4 + T lymphocytes were stimulated in the presence or absence of azathioprine or 6-MP for 3 days, as indicated. GTP-bound Rac1 was analyzed using PAK to determine Rac1 activation. Azathioprine treatment led to a reduction of CD28-dependent Rac1 activation. One representative experiment out of five is shown. ( c ) 6-MP and 6-TG fail to modulate Ras activation. GTP-bound Ras (Ras-GTP) was analyzed using Raf RGD to determine Ras activation. Azathioprine and its metabolites did not affect Ras activation. One representative experiment of three is shown. ( d ) Downregulation of STAT-3 in primary T cells upon treatment with azathioprine or 6-MP. Purified CD4 + ), GTP-bound Rac1 was obtained using PAK, and STAT-3 levels were determined by immunoblotting. ( e ) Competition of GTP binding to Rac1 or Ras by 6-Thio-GTP. Recombinant Rac1 or Ras was incubated with radiolabeled GTP ([ 3 H]GTP) and increasing amounts of chemically synthesized 6-Thio-GTP (0–500 μM). Next, Rac1 was obtained using PAK-1 agarose, followed by analysis of [ 3 H]GTP-bound Rac1 by scintillation counting. Similarly, Ras was obtained using Raf RGD agarose followed by analysis of [ 3 H]GTP-bound Ras by scintillation counting. 6-Thio-GTP led to a concentration-dependent suppression of [ 3 H]GTP-bound Rac1 but had little effect on [ 3 H]GTP-bound Ras.
    Figure Legend Snippet: ( a ) Rac1 activation assay in activated primary T cells. Purified CD4 + T lymphocytes were stimulated with IL-2 and antibodies to CD3 or IL-2 plus antibodies to CD3 and CD28 for 3 days. GTP-bound Rac1 (Rac1-GTP) was analyzed using PAK to determine Rac1 activation. CD28 costimulation led to induction of Rac1 activation in primary T cells. ( b ) Azathioprine and 6-MP suppress Rac1 activation. Purified CD4 + T lymphocytes were stimulated in the presence or absence of azathioprine or 6-MP for 3 days, as indicated. GTP-bound Rac1 was analyzed using PAK to determine Rac1 activation. Azathioprine treatment led to a reduction of CD28-dependent Rac1 activation. One representative experiment out of five is shown. ( c ) 6-MP and 6-TG fail to modulate Ras activation. GTP-bound Ras (Ras-GTP) was analyzed using Raf RGD to determine Ras activation. Azathioprine and its metabolites did not affect Ras activation. One representative experiment of three is shown. ( d ) Downregulation of STAT-3 in primary T cells upon treatment with azathioprine or 6-MP. Purified CD4 + ), GTP-bound Rac1 was obtained using PAK, and STAT-3 levels were determined by immunoblotting. ( e ) Competition of GTP binding to Rac1 or Ras by 6-Thio-GTP. Recombinant Rac1 or Ras was incubated with radiolabeled GTP ([ 3 H]GTP) and increasing amounts of chemically synthesized 6-Thio-GTP (0–500 μM). Next, Rac1 was obtained using PAK-1 agarose, followed by analysis of [ 3 H]GTP-bound Rac1 by scintillation counting. Similarly, Ras was obtained using Raf RGD agarose followed by analysis of [ 3 H]GTP-bound Ras by scintillation counting. 6-Thio-GTP led to a concentration-dependent suppression of [ 3 H]GTP-bound Rac1 but had little effect on [ 3 H]GTP-bound Ras.

    Techniques Used: Activation Assay, Purification, Binding Assay, Recombinant, Incubation, Synthesized, Concentration Assay

    5) Product Images from "T cell receptor-driven transendothelial migration of human effector memory CD4 T cells involves Vav, Rac and Myosin IIA"

    Article Title: T cell receptor-driven transendothelial migration of human effector memory CD4 T cells involves Vav, Rac and Myosin IIA

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1201817

    Vav is required for TCR-driven TEM. A. TEM assays of EM CD4 T cells treated with vehicle and 5 μM 6-Thio GTP for 44 hours. Graphs show %TEM of cells lacking the Vβ2TCR (VB2−, which do not interact with TSST-1 and transmigrate in
    Figure Legend Snippet: Vav is required for TCR-driven TEM. A. TEM assays of EM CD4 T cells treated with vehicle and 5 μM 6-Thio GTP for 44 hours. Graphs show %TEM of cells lacking the Vβ2TCR (VB2−, which do not interact with TSST-1 and transmigrate in

    Techniques Used: Transmission Electron Microscopy

    6) Product Images from "CD28-dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes"

    Article Title: CD28-dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes

    Journal: Journal of Clinical Investigation

    doi: 10.1172/JCI200316432

    ( a ) Rac1 activation assay in activated primary T cells. Purified CD4 + T lymphocytes were stimulated with IL-2 and antibodies to CD3 or IL-2 plus antibodies to CD3 and CD28 for 3 days. GTP-bound Rac1 (Rac1-GTP) was analyzed using PAK to determine Rac1 activation. CD28 costimulation led to induction of Rac1 activation in primary T cells. ( b ) Azathioprine and 6-MP suppress Rac1 activation. Purified CD4 + T lymphocytes were stimulated in the presence or absence of azathioprine or 6-MP for 3 days, as indicated. GTP-bound Rac1 was analyzed using PAK to determine Rac1 activation. Azathioprine treatment led to a reduction of CD28-dependent Rac1 activation. One representative experiment out of five is shown. ( c ) 6-MP and 6-TG fail to modulate Ras activation. GTP-bound Ras (Ras-GTP) was analyzed using Raf RGD to determine Ras activation. Azathioprine and its metabolites did not affect Ras activation. One representative experiment of three is shown. ( d ) Downregulation of STAT-3 in primary T cells upon treatment with azathioprine or 6-MP. Purified CD4 + ), GTP-bound Rac1 was obtained using PAK, and STAT-3 levels were determined by immunoblotting. ( e ) Competition of GTP binding to Rac1 or Ras by 6-Thio-GTP. Recombinant Rac1 or Ras was incubated with radiolabeled GTP ([ 3 H]GTP) and increasing amounts of chemically synthesized 6-Thio-GTP (0–500 μM). Next, Rac1 was obtained using PAK-1 agarose, followed by analysis of [ 3 H]GTP-bound Rac1 by scintillation counting. Similarly, Ras was obtained using Raf RGD agarose followed by analysis of [ 3 H]GTP-bound Ras by scintillation counting. 6-Thio-GTP led to a concentration-dependent suppression of [ 3 H]GTP-bound Rac1 but had little effect on [ 3 H]GTP-bound Ras.
    Figure Legend Snippet: ( a ) Rac1 activation assay in activated primary T cells. Purified CD4 + T lymphocytes were stimulated with IL-2 and antibodies to CD3 or IL-2 plus antibodies to CD3 and CD28 for 3 days. GTP-bound Rac1 (Rac1-GTP) was analyzed using PAK to determine Rac1 activation. CD28 costimulation led to induction of Rac1 activation in primary T cells. ( b ) Azathioprine and 6-MP suppress Rac1 activation. Purified CD4 + T lymphocytes were stimulated in the presence or absence of azathioprine or 6-MP for 3 days, as indicated. GTP-bound Rac1 was analyzed using PAK to determine Rac1 activation. Azathioprine treatment led to a reduction of CD28-dependent Rac1 activation. One representative experiment out of five is shown. ( c ) 6-MP and 6-TG fail to modulate Ras activation. GTP-bound Ras (Ras-GTP) was analyzed using Raf RGD to determine Ras activation. Azathioprine and its metabolites did not affect Ras activation. One representative experiment of three is shown. ( d ) Downregulation of STAT-3 in primary T cells upon treatment with azathioprine or 6-MP. Purified CD4 + ), GTP-bound Rac1 was obtained using PAK, and STAT-3 levels were determined by immunoblotting. ( e ) Competition of GTP binding to Rac1 or Ras by 6-Thio-GTP. Recombinant Rac1 or Ras was incubated with radiolabeled GTP ([ 3 H]GTP) and increasing amounts of chemically synthesized 6-Thio-GTP (0–500 μM). Next, Rac1 was obtained using PAK-1 agarose, followed by analysis of [ 3 H]GTP-bound Rac1 by scintillation counting. Similarly, Ras was obtained using Raf RGD agarose followed by analysis of [ 3 H]GTP-bound Ras by scintillation counting. 6-Thio-GTP led to a concentration-dependent suppression of [ 3 H]GTP-bound Rac1 but had little effect on [ 3 H]GTP-bound Ras.

    Techniques Used: Activation Assay, Purification, Binding Assay, Recombinant, Incubation, Synthesized, Concentration Assay

    7) Product Images from "CD28-dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes"

    Article Title: CD28-dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes

    Journal: Journal of Clinical Investigation

    doi: 10.1172/JCI200316432

    ( a ) Rac1 activation assay in activated primary T cells. Purified CD4 + T lymphocytes were stimulated with IL-2 and antibodies to CD3 or IL-2 plus antibodies to CD3 and CD28 for 3 days. GTP-bound Rac1 (Rac1-GTP) was analyzed using PAK to determine Rac1 activation. CD28 costimulation led to induction of Rac1 activation in primary T cells. ( b ) Azathioprine and 6-MP suppress Rac1 activation. Purified CD4 + T lymphocytes were stimulated in the presence or absence of azathioprine or 6-MP for 3 days, as indicated. GTP-bound Rac1 was analyzed using PAK to determine Rac1 activation. Azathioprine treatment led to a reduction of CD28-dependent Rac1 activation. One representative experiment out of five is shown. ( c ) 6-MP and 6-TG fail to modulate Ras activation. GTP-bound Ras (Ras-GTP) was analyzed using Raf RGD to determine Ras activation. Azathioprine and its metabolites did not affect Ras activation. One representative experiment of three is shown. ( d ) Downregulation of STAT-3 in primary T cells upon treatment with azathioprine or 6-MP. Purified CD4 + ), GTP-bound Rac1 was obtained using PAK, and STAT-3 levels were determined by immunoblotting. ( e ) Competition of GTP binding to Rac1 or Ras by 6-Thio-GTP. Recombinant Rac1 or Ras was incubated with radiolabeled GTP ([ 3 H]GTP) and increasing amounts of chemically synthesized 6-Thio-GTP (0–500 μM). Next, Rac1 was obtained using PAK-1 agarose, followed by analysis of [ 3 H]GTP-bound Rac1 by scintillation counting. Similarly, Ras was obtained using Raf RGD agarose followed by analysis of [ 3 H]GTP-bound Ras by scintillation counting. 6-Thio-GTP led to a concentration-dependent suppression of [ 3 H]GTP-bound Rac1 but had little effect on [ 3 H]GTP-bound Ras.
    Figure Legend Snippet: ( a ) Rac1 activation assay in activated primary T cells. Purified CD4 + T lymphocytes were stimulated with IL-2 and antibodies to CD3 or IL-2 plus antibodies to CD3 and CD28 for 3 days. GTP-bound Rac1 (Rac1-GTP) was analyzed using PAK to determine Rac1 activation. CD28 costimulation led to induction of Rac1 activation in primary T cells. ( b ) Azathioprine and 6-MP suppress Rac1 activation. Purified CD4 + T lymphocytes were stimulated in the presence or absence of azathioprine or 6-MP for 3 days, as indicated. GTP-bound Rac1 was analyzed using PAK to determine Rac1 activation. Azathioprine treatment led to a reduction of CD28-dependent Rac1 activation. One representative experiment out of five is shown. ( c ) 6-MP and 6-TG fail to modulate Ras activation. GTP-bound Ras (Ras-GTP) was analyzed using Raf RGD to determine Ras activation. Azathioprine and its metabolites did not affect Ras activation. One representative experiment of three is shown. ( d ) Downregulation of STAT-3 in primary T cells upon treatment with azathioprine or 6-MP. Purified CD4 + ), GTP-bound Rac1 was obtained using PAK, and STAT-3 levels were determined by immunoblotting. ( e ) Competition of GTP binding to Rac1 or Ras by 6-Thio-GTP. Recombinant Rac1 or Ras was incubated with radiolabeled GTP ([ 3 H]GTP) and increasing amounts of chemically synthesized 6-Thio-GTP (0–500 μM). Next, Rac1 was obtained using PAK-1 agarose, followed by analysis of [ 3 H]GTP-bound Rac1 by scintillation counting. Similarly, Ras was obtained using Raf RGD agarose followed by analysis of [ 3 H]GTP-bound Ras by scintillation counting. 6-Thio-GTP led to a concentration-dependent suppression of [ 3 H]GTP-bound Rac1 but had little effect on [ 3 H]GTP-bound Ras.

    Techniques Used: Activation Assay, Purification, Binding Assay, Recombinant, Incubation, Synthesized, Concentration Assay

    8) Product Images from "The Role of Rho-GTPases and actin polymerization during Macrophage Tunneling Nanotube Biogenesis"

    Article Title: The Role of Rho-GTPases and actin polymerization during Macrophage Tunneling Nanotube Biogenesis

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-08950-7

    Roles of Cdc42 and Rac1 during TNT formation. ( a ) Following quick adherence, GFP-CAAX RAW/LR5 macrophages were incubated with growth media containing vehicle control, Cdc42 inhibitor ML-141 (10 μM) or Rac1 inhibitor 6-thio-GTP (10 μM). Time-lapse live imaging of GFP-CAAX RAW/LR5 macrophages in the presence of DMSO (upper panels), Cdc42 (middle) or Rac1 (bottom) inhibitors in BWD buffer imaged over 10 min periods. Arrow heads indicate when ‘TNT-like’ protrusions initiate. Scale bar: 5 µm. ( b ) Quantitation of the number of TNT-like protrusions ( > 8 µm in length) per cell. ( c ) Quantitation of the average duration of TNT-like protrusions ( > 8 μm) over the course of the time-lapse images (10 min). ( d ) Quantitation of the maximum length of TNT-like protrusions and ( e ) the TNT-like protrusion rate (maximum length/time it takes to reach the maximum length). Data represents the mean average of at least 25 cells. Error bars +/−SEM with *p
    Figure Legend Snippet: Roles of Cdc42 and Rac1 during TNT formation. ( a ) Following quick adherence, GFP-CAAX RAW/LR5 macrophages were incubated with growth media containing vehicle control, Cdc42 inhibitor ML-141 (10 μM) or Rac1 inhibitor 6-thio-GTP (10 μM). Time-lapse live imaging of GFP-CAAX RAW/LR5 macrophages in the presence of DMSO (upper panels), Cdc42 (middle) or Rac1 (bottom) inhibitors in BWD buffer imaged over 10 min periods. Arrow heads indicate when ‘TNT-like’ protrusions initiate. Scale bar: 5 µm. ( b ) Quantitation of the number of TNT-like protrusions ( > 8 µm in length) per cell. ( c ) Quantitation of the average duration of TNT-like protrusions ( > 8 μm) over the course of the time-lapse images (10 min). ( d ) Quantitation of the maximum length of TNT-like protrusions and ( e ) the TNT-like protrusion rate (maximum length/time it takes to reach the maximum length). Data represents the mean average of at least 25 cells. Error bars +/−SEM with *p

    Techniques Used: Incubation, Imaging, Quantitation Assay

    Both the WASP pathway and the Rac1 pathway contribute to TNT formation. ( a ) Control RAW/LR5 macrophages and shWASP cells were incubated in growth media containing vehicle control or the Cdc42 inhibitor ML-141 (10 μM). After 4 hours the number of TNT connections formed was quantified. ( b ) Control RAW/LR5 macrophages and shWASP cells were incubated in growth media containing vehicle control (black bars) or the Rac1 inhibitor 6-thio-GTP (10 μM) (gray pattern bars). After 4 hours the number of TNT connections formed was quantified. ( c ) RAW/LR5 macrophages were transiently transfected with either non-targeting or shRNA constructs against WASP or WAVE proteins overnight and then selected with puromycin. Representative western blot analysis of the relative expression levels of WASP (left) and WAVE (right); β-actin is used as a loading control. Selected cells were plated in MatTek dishes and TNTs were allowed to form overnight and then quantified and plotted as in previous figures for at least 3 independent experiments. Error bars +/−SEM with *p
    Figure Legend Snippet: Both the WASP pathway and the Rac1 pathway contribute to TNT formation. ( a ) Control RAW/LR5 macrophages and shWASP cells were incubated in growth media containing vehicle control or the Cdc42 inhibitor ML-141 (10 μM). After 4 hours the number of TNT connections formed was quantified. ( b ) Control RAW/LR5 macrophages and shWASP cells were incubated in growth media containing vehicle control (black bars) or the Rac1 inhibitor 6-thio-GTP (10 μM) (gray pattern bars). After 4 hours the number of TNT connections formed was quantified. ( c ) RAW/LR5 macrophages were transiently transfected with either non-targeting or shRNA constructs against WASP or WAVE proteins overnight and then selected with puromycin. Representative western blot analysis of the relative expression levels of WASP (left) and WAVE (right); β-actin is used as a loading control. Selected cells were plated in MatTek dishes and TNTs were allowed to form overnight and then quantified and plotted as in previous figures for at least 3 independent experiments. Error bars +/−SEM with *p

    Techniques Used: Incubation, Transfection, shRNA, Construct, Western Blot, Expressing

    Actin polymerization signaling pathways are required for TNT formation in RAW/LR5 macrophages. ( a ) Montage of time-lapse live images of GFP-CAAX RAW/LR5 macrophages showing the formation of a TNT-like protrusion (indicated by the yellow arrow) that then connects to an adjacent cell. For visualization of fine less intense structures within the narrow dynamic range available for display, images were local contrast enhanced. Scale bar: 10 µm. ( b ) Following quick adherence, RAW/LR5 macrophages were incubated with growth media containing vehicle control, actin polymerization inhibitor cytochalasin D (2 μM), or microtubule polymerization inhibitor Nocodazole (2 μM). TNT formation was quantified 4 hours after treatment. The number of TNT connections is represented as in (2d). ( c ) Following quick adherence, RAW/LR5 macrophages were incubated with growth media containing vehicle control, Arp2/3 inhibitor CK666 (40 μM), Cdc42 inhibitor ML-141 (10 μM) or Rac1 inhibitor 6-thio-GTP (10 μM). TNT formation was quantified 4 hours after treatment. ( d ) Following quick adherence, bone marrow derived macrophages (BMMs) isolated from 3 independent mice were treated with vehicle control, Arp2/3 inhibitor CK666 (40 μM), or actin polymerization inhibitor cytochalasin D (2 μM). TNT formation was quantified 4 hours after treatment. ( e ) Following quick adherence, BMMs isolated from 3 independent mice were treated with vehicle control, Cdc42 inhibitor ML-141 (10 μM) or Rac1 inhibitor 6-thio-GTP (10μM). TNT formation was quantified 4 hours after treatment. Data in all graphs is represented as dot plots showing the individual values of the number of TNT connections for each independent experiment in each case. The outlined histograms represent the mean average of at least 3 independent experiments. Error bars +/−SEM with *p
    Figure Legend Snippet: Actin polymerization signaling pathways are required for TNT formation in RAW/LR5 macrophages. ( a ) Montage of time-lapse live images of GFP-CAAX RAW/LR5 macrophages showing the formation of a TNT-like protrusion (indicated by the yellow arrow) that then connects to an adjacent cell. For visualization of fine less intense structures within the narrow dynamic range available for display, images were local contrast enhanced. Scale bar: 10 µm. ( b ) Following quick adherence, RAW/LR5 macrophages were incubated with growth media containing vehicle control, actin polymerization inhibitor cytochalasin D (2 μM), or microtubule polymerization inhibitor Nocodazole (2 μM). TNT formation was quantified 4 hours after treatment. The number of TNT connections is represented as in (2d). ( c ) Following quick adherence, RAW/LR5 macrophages were incubated with growth media containing vehicle control, Arp2/3 inhibitor CK666 (40 μM), Cdc42 inhibitor ML-141 (10 μM) or Rac1 inhibitor 6-thio-GTP (10 μM). TNT formation was quantified 4 hours after treatment. ( d ) Following quick adherence, bone marrow derived macrophages (BMMs) isolated from 3 independent mice were treated with vehicle control, Arp2/3 inhibitor CK666 (40 μM), or actin polymerization inhibitor cytochalasin D (2 μM). TNT formation was quantified 4 hours after treatment. ( e ) Following quick adherence, BMMs isolated from 3 independent mice were treated with vehicle control, Cdc42 inhibitor ML-141 (10 μM) or Rac1 inhibitor 6-thio-GTP (10μM). TNT formation was quantified 4 hours after treatment. Data in all graphs is represented as dot plots showing the individual values of the number of TNT connections for each independent experiment in each case. The outlined histograms represent the mean average of at least 3 independent experiments. Error bars +/−SEM with *p

    Techniques Used: Incubation, Derivative Assay, Isolation, Mouse Assay

    9) Product Images from "The Role of Rho-GTPases and actin polymerization during Macrophage Tunneling Nanotube Biogenesis"

    Article Title: The Role of Rho-GTPases and actin polymerization during Macrophage Tunneling Nanotube Biogenesis

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-08950-7

    Roles of Cdc42 and Rac1 during TNT formation. ( a ) Following quick adherence, GFP-CAAX RAW/LR5 macrophages were incubated with growth media containing vehicle control, Cdc42 inhibitor ML-141 (10 μM) or Rac1 inhibitor 6-thio-GTP (10 μM). Time-lapse live imaging of GFP-CAAX RAW/LR5 macrophages in the presence of DMSO (upper panels), Cdc42 (middle) or Rac1 (bottom) inhibitors in BWD buffer imaged over 10 min periods. Arrow heads indicate when ‘TNT-like’ protrusions initiate. Scale bar: 5 µm. ( b ) Quantitation of the number of TNT-like protrusions ( > 8 µm in length) per cell. ( c ) Quantitation of the average duration of TNT-like protrusions ( > 8 μm) over the course of the time-lapse images (10 min). ( d ) Quantitation of the maximum length of TNT-like protrusions and ( e ) the TNT-like protrusion rate (maximum length/time it takes to reach the maximum length). Data represents the mean average of at least 25 cells. Error bars +/−SEM with *p
    Figure Legend Snippet: Roles of Cdc42 and Rac1 during TNT formation. ( a ) Following quick adherence, GFP-CAAX RAW/LR5 macrophages were incubated with growth media containing vehicle control, Cdc42 inhibitor ML-141 (10 μM) or Rac1 inhibitor 6-thio-GTP (10 μM). Time-lapse live imaging of GFP-CAAX RAW/LR5 macrophages in the presence of DMSO (upper panels), Cdc42 (middle) or Rac1 (bottom) inhibitors in BWD buffer imaged over 10 min periods. Arrow heads indicate when ‘TNT-like’ protrusions initiate. Scale bar: 5 µm. ( b ) Quantitation of the number of TNT-like protrusions ( > 8 µm in length) per cell. ( c ) Quantitation of the average duration of TNT-like protrusions ( > 8 μm) over the course of the time-lapse images (10 min). ( d ) Quantitation of the maximum length of TNT-like protrusions and ( e ) the TNT-like protrusion rate (maximum length/time it takes to reach the maximum length). Data represents the mean average of at least 25 cells. Error bars +/−SEM with *p

    Techniques Used: Incubation, Imaging, Quantitation Assay

    Both the WASP pathway and the Rac1 pathway contribute to TNT formation. ( a ) Control RAW/LR5 macrophages and shWASP cells were incubated in growth media containing vehicle control or the Cdc42 inhibitor ML-141 (10 μM). After 4 hours the number of TNT connections formed was quantified. ( b ) Control RAW/LR5 macrophages and shWASP cells were incubated in growth media containing vehicle control (black bars) or the Rac1 inhibitor 6-thio-GTP (10 μM) (gray pattern bars). After 4 hours the number of TNT connections formed was quantified. ( c ) RAW/LR5 macrophages were transiently transfected with either non-targeting or shRNA constructs against WASP or WAVE proteins overnight and then selected with puromycin. Representative western blot analysis of the relative expression levels of WASP (left) and WAVE (right); β-actin is used as a loading control. Selected cells were plated in MatTek dishes and TNTs were allowed to form overnight and then quantified and plotted as in previous figures for at least 3 independent experiments. Error bars +/−SEM with *p
    Figure Legend Snippet: Both the WASP pathway and the Rac1 pathway contribute to TNT formation. ( a ) Control RAW/LR5 macrophages and shWASP cells were incubated in growth media containing vehicle control or the Cdc42 inhibitor ML-141 (10 μM). After 4 hours the number of TNT connections formed was quantified. ( b ) Control RAW/LR5 macrophages and shWASP cells were incubated in growth media containing vehicle control (black bars) or the Rac1 inhibitor 6-thio-GTP (10 μM) (gray pattern bars). After 4 hours the number of TNT connections formed was quantified. ( c ) RAW/LR5 macrophages were transiently transfected with either non-targeting or shRNA constructs against WASP or WAVE proteins overnight and then selected with puromycin. Representative western blot analysis of the relative expression levels of WASP (left) and WAVE (right); β-actin is used as a loading control. Selected cells were plated in MatTek dishes and TNTs were allowed to form overnight and then quantified and plotted as in previous figures for at least 3 independent experiments. Error bars +/−SEM with *p

    Techniques Used: Incubation, Transfection, shRNA, Construct, Western Blot, Expressing

    Actin polymerization signaling pathways are required for TNT formation in RAW/LR5 macrophages. ( a ) Montage of time-lapse live images of GFP-CAAX RAW/LR5 macrophages showing the formation of a TNT-like protrusion (indicated by the yellow arrow) that then connects to an adjacent cell. For visualization of fine less intense structures within the narrow dynamic range available for display, images were local contrast enhanced. Scale bar: 10 µm. ( b ) Following quick adherence, RAW/LR5 macrophages were incubated with growth media containing vehicle control, actin polymerization inhibitor cytochalasin D (2 μM), or microtubule polymerization inhibitor Nocodazole (2 μM). TNT formation was quantified 4 hours after treatment. The number of TNT connections is represented as in (2d). ( c ) Following quick adherence, RAW/LR5 macrophages were incubated with growth media containing vehicle control, Arp2/3 inhibitor CK666 (40 μM), Cdc42 inhibitor ML-141 (10 μM) or Rac1 inhibitor 6-thio-GTP (10 μM). TNT formation was quantified 4 hours after treatment. ( d ) Following quick adherence, bone marrow derived macrophages (BMMs) isolated from 3 independent mice were treated with vehicle control, Arp2/3 inhibitor CK666 (40 μM), or actin polymerization inhibitor cytochalasin D (2 μM). TNT formation was quantified 4 hours after treatment. ( e ) Following quick adherence, BMMs isolated from 3 independent mice were treated with vehicle control, Cdc42 inhibitor ML-141 (10 μM) or Rac1 inhibitor 6-thio-GTP (10μM). TNT formation was quantified 4 hours after treatment. Data in all graphs is represented as dot plots showing the individual values of the number of TNT connections for each independent experiment in each case. The outlined histograms represent the mean average of at least 3 independent experiments. Error bars +/−SEM with *p
    Figure Legend Snippet: Actin polymerization signaling pathways are required for TNT formation in RAW/LR5 macrophages. ( a ) Montage of time-lapse live images of GFP-CAAX RAW/LR5 macrophages showing the formation of a TNT-like protrusion (indicated by the yellow arrow) that then connects to an adjacent cell. For visualization of fine less intense structures within the narrow dynamic range available for display, images were local contrast enhanced. Scale bar: 10 µm. ( b ) Following quick adherence, RAW/LR5 macrophages were incubated with growth media containing vehicle control, actin polymerization inhibitor cytochalasin D (2 μM), or microtubule polymerization inhibitor Nocodazole (2 μM). TNT formation was quantified 4 hours after treatment. The number of TNT connections is represented as in (2d). ( c ) Following quick adherence, RAW/LR5 macrophages were incubated with growth media containing vehicle control, Arp2/3 inhibitor CK666 (40 μM), Cdc42 inhibitor ML-141 (10 μM) or Rac1 inhibitor 6-thio-GTP (10 μM). TNT formation was quantified 4 hours after treatment. ( d ) Following quick adherence, bone marrow derived macrophages (BMMs) isolated from 3 independent mice were treated with vehicle control, Arp2/3 inhibitor CK666 (40 μM), or actin polymerization inhibitor cytochalasin D (2 μM). TNT formation was quantified 4 hours after treatment. ( e ) Following quick adherence, BMMs isolated from 3 independent mice were treated with vehicle control, Cdc42 inhibitor ML-141 (10 μM) or Rac1 inhibitor 6-thio-GTP (10μM). TNT formation was quantified 4 hours after treatment. Data in all graphs is represented as dot plots showing the individual values of the number of TNT connections for each independent experiment in each case. The outlined histograms represent the mean average of at least 3 independent experiments. Error bars +/−SEM with *p

    Techniques Used: Incubation, Derivative Assay, Isolation, Mouse Assay

    10) Product Images from "CD28-dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes"

    Article Title: CD28-dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes

    Journal: Journal of Clinical Investigation

    doi: 10.1172/JCI200316432

    ( a ) Rac1 activation assay in activated primary T cells. Purified CD4 + T lymphocytes were stimulated with IL-2 and antibodies to CD3 or IL-2 plus antibodies to CD3 and CD28 for 3 days. GTP-bound Rac1 (Rac1-GTP) was analyzed using PAK to determine Rac1 activation. CD28 costimulation led to induction of Rac1 activation in primary T cells. ( b ) Azathioprine and 6-MP suppress Rac1 activation. Purified CD4 + T lymphocytes were stimulated in the presence or absence of azathioprine or 6-MP for 3 days, as indicated. GTP-bound Rac1 was analyzed using PAK to determine Rac1 activation. Azathioprine treatment led to a reduction of CD28-dependent Rac1 activation. One representative experiment out of five is shown. ( c ) 6-MP and 6-TG fail to modulate Ras activation. GTP-bound Ras (Ras-GTP) was analyzed using Raf RGD to determine Ras activation. Azathioprine and its metabolites did not affect Ras activation. One representative experiment of three is shown. ( d ) Downregulation of STAT-3 in primary T cells upon treatment with azathioprine or 6-MP. Purified CD4 + ), GTP-bound Rac1 was obtained using PAK, and STAT-3 levels were determined by immunoblotting. ( e ) Competition of GTP binding to Rac1 or Ras by 6-Thio-GTP. Recombinant Rac1 or Ras was incubated with radiolabeled GTP ([ 3 H]GTP) and increasing amounts of chemically synthesized 6-Thio-GTP (0–500 μM). Next, Rac1 was obtained using PAK-1 agarose, followed by analysis of [ 3 H]GTP-bound Rac1 by scintillation counting. Similarly, Ras was obtained using Raf RGD agarose followed by analysis of [ 3 H]GTP-bound Ras by scintillation counting. 6-Thio-GTP led to a concentration-dependent suppression of [ 3 H]GTP-bound Rac1 but had little effect on [ 3 H]GTP-bound Ras.
    Figure Legend Snippet: ( a ) Rac1 activation assay in activated primary T cells. Purified CD4 + T lymphocytes were stimulated with IL-2 and antibodies to CD3 or IL-2 plus antibodies to CD3 and CD28 for 3 days. GTP-bound Rac1 (Rac1-GTP) was analyzed using PAK to determine Rac1 activation. CD28 costimulation led to induction of Rac1 activation in primary T cells. ( b ) Azathioprine and 6-MP suppress Rac1 activation. Purified CD4 + T lymphocytes were stimulated in the presence or absence of azathioprine or 6-MP for 3 days, as indicated. GTP-bound Rac1 was analyzed using PAK to determine Rac1 activation. Azathioprine treatment led to a reduction of CD28-dependent Rac1 activation. One representative experiment out of five is shown. ( c ) 6-MP and 6-TG fail to modulate Ras activation. GTP-bound Ras (Ras-GTP) was analyzed using Raf RGD to determine Ras activation. Azathioprine and its metabolites did not affect Ras activation. One representative experiment of three is shown. ( d ) Downregulation of STAT-3 in primary T cells upon treatment with azathioprine or 6-MP. Purified CD4 + ), GTP-bound Rac1 was obtained using PAK, and STAT-3 levels were determined by immunoblotting. ( e ) Competition of GTP binding to Rac1 or Ras by 6-Thio-GTP. Recombinant Rac1 or Ras was incubated with radiolabeled GTP ([ 3 H]GTP) and increasing amounts of chemically synthesized 6-Thio-GTP (0–500 μM). Next, Rac1 was obtained using PAK-1 agarose, followed by analysis of [ 3 H]GTP-bound Rac1 by scintillation counting. Similarly, Ras was obtained using Raf RGD agarose followed by analysis of [ 3 H]GTP-bound Ras by scintillation counting. 6-Thio-GTP led to a concentration-dependent suppression of [ 3 H]GTP-bound Rac1 but had little effect on [ 3 H]GTP-bound Ras.

    Techniques Used: Activation Assay, Purification, Binding Assay, Recombinant, Incubation, Synthesized, Concentration Assay

    11) Product Images from "T cell receptor-driven transendothelial migration of human effector memory CD4 T cells involves Vav, Rac and Myosin IIA"

    Article Title: T cell receptor-driven transendothelial migration of human effector memory CD4 T cells involves Vav, Rac and Myosin IIA

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1201817

    Vav is required for TCR-driven TEM. A. TEM assays of EM CD4 T cells treated with vehicle and 5 μM 6-Thio GTP for 44 hours. Graphs show %TEM of cells lacking the Vβ2TCR (VB2−, which do not interact with TSST-1 and transmigrate in
    Figure Legend Snippet: Vav is required for TCR-driven TEM. A. TEM assays of EM CD4 T cells treated with vehicle and 5 μM 6-Thio GTP for 44 hours. Graphs show %TEM of cells lacking the Vβ2TCR (VB2−, which do not interact with TSST-1 and transmigrate in

    Techniques Used: Transmission Electron Microscopy

    12) Product Images from "CD28-dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes"

    Article Title: CD28-dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes

    Journal: Journal of Clinical Investigation

    doi: 10.1172/JCI200316432

    ( a ) Rac1 activation assay in activated primary T cells. Purified CD4 + T lymphocytes were stimulated with IL-2 and antibodies to CD3 or IL-2 plus antibodies to CD3 and CD28 for 3 days. GTP-bound Rac1 (Rac1-GTP) was analyzed using PAK to determine Rac1 activation. CD28 costimulation led to induction of Rac1 activation in primary T cells. ( b ) Azathioprine and 6-MP suppress Rac1 activation. Purified CD4 + T lymphocytes were stimulated in the presence or absence of azathioprine or 6-MP for 3 days, as indicated. GTP-bound Rac1 was analyzed using PAK to determine Rac1 activation. Azathioprine treatment led to a reduction of CD28-dependent Rac1 activation. One representative experiment out of five is shown. ( c ) 6-MP and 6-TG fail to modulate Ras activation. GTP-bound Ras (Ras-GTP) was analyzed using Raf RGD to determine Ras activation. Azathioprine and its metabolites did not affect Ras activation. One representative experiment of three is shown. ( d ) Downregulation of STAT-3 in primary T cells upon treatment with azathioprine or 6-MP. Purified CD4 + ), GTP-bound Rac1 was obtained using PAK, and STAT-3 levels were determined by immunoblotting. ( e ) Competition of GTP binding to Rac1 or Ras by 6-Thio-GTP. Recombinant Rac1 or Ras was incubated with radiolabeled GTP ([ 3 H]GTP) and increasing amounts of chemically synthesized 6-Thio-GTP (0–500 μM). Next, Rac1 was obtained using PAK-1 agarose, followed by analysis of [ 3 H]GTP-bound Rac1 by scintillation counting. Similarly, Ras was obtained using Raf RGD agarose followed by analysis of [ 3 H]GTP-bound Ras by scintillation counting. 6-Thio-GTP led to a concentration-dependent suppression of [ 3 H]GTP-bound Rac1 but had little effect on [ 3 H]GTP-bound Ras.
    Figure Legend Snippet: ( a ) Rac1 activation assay in activated primary T cells. Purified CD4 + T lymphocytes were stimulated with IL-2 and antibodies to CD3 or IL-2 plus antibodies to CD3 and CD28 for 3 days. GTP-bound Rac1 (Rac1-GTP) was analyzed using PAK to determine Rac1 activation. CD28 costimulation led to induction of Rac1 activation in primary T cells. ( b ) Azathioprine and 6-MP suppress Rac1 activation. Purified CD4 + T lymphocytes were stimulated in the presence or absence of azathioprine or 6-MP for 3 days, as indicated. GTP-bound Rac1 was analyzed using PAK to determine Rac1 activation. Azathioprine treatment led to a reduction of CD28-dependent Rac1 activation. One representative experiment out of five is shown. ( c ) 6-MP and 6-TG fail to modulate Ras activation. GTP-bound Ras (Ras-GTP) was analyzed using Raf RGD to determine Ras activation. Azathioprine and its metabolites did not affect Ras activation. One representative experiment of three is shown. ( d ) Downregulation of STAT-3 in primary T cells upon treatment with azathioprine or 6-MP. Purified CD4 + ), GTP-bound Rac1 was obtained using PAK, and STAT-3 levels were determined by immunoblotting. ( e ) Competition of GTP binding to Rac1 or Ras by 6-Thio-GTP. Recombinant Rac1 or Ras was incubated with radiolabeled GTP ([ 3 H]GTP) and increasing amounts of chemically synthesized 6-Thio-GTP (0–500 μM). Next, Rac1 was obtained using PAK-1 agarose, followed by analysis of [ 3 H]GTP-bound Rac1 by scintillation counting. Similarly, Ras was obtained using Raf RGD agarose followed by analysis of [ 3 H]GTP-bound Ras by scintillation counting. 6-Thio-GTP led to a concentration-dependent suppression of [ 3 H]GTP-bound Rac1 but had little effect on [ 3 H]GTP-bound Ras.

    Techniques Used: Activation Assay, Purification, Binding Assay, Recombinant, Incubation, Synthesized, Concentration Assay

    Related Articles

    Flow Cytometry:

    Article Title: T cell receptor-driven transendothelial migration of human effector memory CD4 T cells involves Vav, Rac and Myosin IIA
    Article Snippet: .. Where indicated, EM CD4 T cells were treated with piceatannol (Calbiochem) and Syk inhibitor II (Calbiochem) to inhibit ZAP-70, with, blebbistatin (Sigma) to inhibit myosin IIA, ML-7 (Calbiochem) to inhibit myosin light chain kinase (MLCK), EHop-016 (Calbiochem) to inhibit Rac and Cdc42, 6-Thio-GTP (Jena Bioscience) to inhibit Vav, or vehicle control (DMSO) and then washed prior to flow TEM assays. .. Antibodies used to stain the samples are as follows: anti-Vβ2TCR mAb (Meridian Life Science), anti-NFAT mAb (Becton Dickenson), anti-Phospho-ZAP-70(Tyr319) rabbit polyclonal antibody (Cell Signaling), anti-CD18 mAb, clone MEM-148 (Enzo Life Sciences), anti-Phospho LAT (Tyr191) rabbit polyclonal antibody (Cell Signaling), anti-talin mAb clone 8d4 (Sigma), anti-RACK1 mAb (Santa Cruz Biotechnology).

    Recombinant:

    Article Title: CD28-dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes
    Article Snippet: .. Furthermore, 6-Thio-GTP did not significantly compete with GTP for recombinant Ras, suggesting that 6-Thio-GTP may bind to selected GTPases only. .. Taken together, these data suggest that azathioprine and its metabolites target CD28-mediated signaling in primary T cells by specifically suppressing the activation of the GTPase Rac1 through 6-Thio-GTP, leading to a mitochondrial pathway of apoptosis (Figure ).

    Article Title: CD28-dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes
    Article Snippet: .. Consistently, 6-Thio-GTP was able to bind to recombinant Rac1 under in vitro conditions (although with lower affinity than GTP) (Figure e). .. Furthermore, 6-Thio-GTP did not significantly compete with GTP for recombinant Ras, suggesting that 6-Thio-GTP may bind to selected GTPases only.

    In Vitro:

    Article Title: CD28-dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes
    Article Snippet: .. Consistently, 6-Thio-GTP was able to bind to recombinant Rac1 under in vitro conditions (although with lower affinity than GTP) (Figure e). .. Furthermore, 6-Thio-GTP did not significantly compete with GTP for recombinant Ras, suggesting that 6-Thio-GTP may bind to selected GTPases only.

    Synthesized:

    Article Title: CD28-dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes
    Article Snippet: .. To analyze the capacity of 6-Thio-GTP to bind to Rac1 or Ras, chemically synthesized 6-Thio-GTP (obtained from Jena Bioscience, Jena, Germany) was used. .. Recombinant Rac1 or Ras was incubated with radiolabeled GTP ([3 H]GTP, Amersham) and increasing amounts of 6-Thio-GTP (0–500 μM) followed by Rac1 and Ras pull-down through PAK and Raf RGD and analysis of [3 H]GTP-bound Rac1 or Ras by scintillation counting.

    Activation Assay:

    Article Title: CD28-dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes
    Article Snippet: .. Although the precise mechanism by which binding of 6-Thio-GTP suppresses Rac1 activation in T cells remains to be determined, the function of another GTPase (Ras) was not inhibited by azathioprine and its metabolites. .. Our data are thus consistent with a model in which the specificity of the 6-Thio-GTP–induced blockade of Rac1 function is related to the structure of the Rac1 protein.

    Article Title: T cell receptor-driven transendothelial migration of human effector memory CD4 T cells involves Vav, Rac and Myosin IIA
    Article Snippet: .. Evidence in support of this concept has been provided by studies of the effects of azathioprine, a widely used immunosuppressant whose mode of action has been elucidated relatively recently; azathioprine is metabolized to 6-Thio-GTP and inhibits Vav GEF-mediated activation of Rac in T cells ( ). .. An interpretation of these findings has been that azathioprine inhibits activation of naïve T cells.

    Article Title: CD28-dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes
    Article Snippet: .. Taken together, these data suggest that azathioprine and its metabolites target CD28-mediated signaling in primary T cells by specifically suppressing the activation of the GTPase Rac1 through 6-Thio-GTP, leading to a mitochondrial pathway of apoptosis (Figure ). .. In the present study, we have identified a unique and unexpected role for azathioprine and its metabolites in the control of T cell apoptosis by modulation of Rac1 activation upon CD28 costimulation.

    Binding Assay:

    Article Title: CD28-dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes
    Article Snippet: .. Although the precise mechanism by which binding of 6-Thio-GTP suppresses Rac1 activation in T cells remains to be determined, the function of another GTPase (Ras) was not inhibited by azathioprine and its metabolites. .. Our data are thus consistent with a model in which the specificity of the 6-Thio-GTP–induced blockade of Rac1 function is related to the structure of the Rac1 protein.

    Transmission Electron Microscopy:

    Article Title: T cell receptor-driven transendothelial migration of human effector memory CD4 T cells involves Vav, Rac and Myosin IIA
    Article Snippet: .. Where indicated, EM CD4 T cells were treated with piceatannol (Calbiochem) and Syk inhibitor II (Calbiochem) to inhibit ZAP-70, with, blebbistatin (Sigma) to inhibit myosin IIA, ML-7 (Calbiochem) to inhibit myosin light chain kinase (MLCK), EHop-016 (Calbiochem) to inhibit Rac and Cdc42, 6-Thio-GTP (Jena Bioscience) to inhibit Vav, or vehicle control (DMSO) and then washed prior to flow TEM assays. .. Antibodies used to stain the samples are as follows: anti-Vβ2TCR mAb (Meridian Life Science), anti-NFAT mAb (Becton Dickenson), anti-Phospho-ZAP-70(Tyr319) rabbit polyclonal antibody (Cell Signaling), anti-CD18 mAb, clone MEM-148 (Enzo Life Sciences), anti-Phospho LAT (Tyr191) rabbit polyclonal antibody (Cell Signaling), anti-talin mAb clone 8d4 (Sigma), anti-RACK1 mAb (Santa Cruz Biotechnology).

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    Jena Bioscience 6 thio gtp
    ( a ) Rac1 activation assay in activated primary T cells. Purified CD4 + T lymphocytes were stimulated with IL-2 and antibodies to CD3 or IL-2 plus antibodies to CD3 and CD28 for 3 days. <t>GTP-bound</t> Rac1 (Rac1-GTP) was analyzed using PAK to determine Rac1 activation. CD28 costimulation led to induction of Rac1 activation in primary T cells. ( b ) Azathioprine and 6-MP suppress Rac1 activation. Purified CD4 + T lymphocytes were stimulated in the presence or absence of azathioprine or 6-MP for 3 days, as indicated. GTP-bound Rac1 was analyzed using PAK to determine Rac1 activation. Azathioprine treatment led to a reduction of CD28-dependent Rac1 activation. One representative experiment out of five is shown. ( c ) 6-MP and 6-TG fail to modulate Ras activation. GTP-bound Ras (Ras-GTP) was analyzed using Raf RGD to determine Ras activation. Azathioprine and its metabolites did not affect Ras activation. One representative experiment of three is shown. ( d ) Downregulation of STAT-3 in primary T cells upon treatment with azathioprine or 6-MP. Purified CD4 + ), GTP-bound Rac1 was obtained using PAK, and STAT-3 levels were determined by immunoblotting. ( e ) Competition of GTP binding to Rac1 or Ras by <t>6-Thio-GTP.</t> Recombinant Rac1 or Ras was incubated with radiolabeled GTP ([ 3 H]GTP) and increasing amounts of chemically synthesized 6-Thio-GTP (0–500 μM). Next, Rac1 was obtained using PAK-1 agarose, followed by analysis of [ 3 H]GTP-bound Rac1 by scintillation counting. Similarly, Ras was obtained using Raf RGD agarose followed by analysis of [ 3 H]GTP-bound Ras by scintillation counting. 6-Thio-GTP led to a concentration-dependent suppression of [ 3 H]GTP-bound Rac1 but had little effect on [ 3 H]GTP-bound Ras.
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    ( a ) Rac1 activation assay in activated primary T cells. Purified CD4 + T lymphocytes were stimulated with IL-2 and antibodies to CD3 or IL-2 plus antibodies to CD3 and CD28 for 3 days. GTP-bound Rac1 (Rac1-GTP) was analyzed using PAK to determine Rac1 activation. CD28 costimulation led to induction of Rac1 activation in primary T cells. ( b ) Azathioprine and 6-MP suppress Rac1 activation. Purified CD4 + T lymphocytes were stimulated in the presence or absence of azathioprine or 6-MP for 3 days, as indicated. GTP-bound Rac1 was analyzed using PAK to determine Rac1 activation. Azathioprine treatment led to a reduction of CD28-dependent Rac1 activation. One representative experiment out of five is shown. ( c ) 6-MP and 6-TG fail to modulate Ras activation. GTP-bound Ras (Ras-GTP) was analyzed using Raf RGD to determine Ras activation. Azathioprine and its metabolites did not affect Ras activation. One representative experiment of three is shown. ( d ) Downregulation of STAT-3 in primary T cells upon treatment with azathioprine or 6-MP. Purified CD4 + ), GTP-bound Rac1 was obtained using PAK, and STAT-3 levels were determined by immunoblotting. ( e ) Competition of GTP binding to Rac1 or Ras by 6-Thio-GTP. Recombinant Rac1 or Ras was incubated with radiolabeled GTP ([ 3 H]GTP) and increasing amounts of chemically synthesized 6-Thio-GTP (0–500 μM). Next, Rac1 was obtained using PAK-1 agarose, followed by analysis of [ 3 H]GTP-bound Rac1 by scintillation counting. Similarly, Ras was obtained using Raf RGD agarose followed by analysis of [ 3 H]GTP-bound Ras by scintillation counting. 6-Thio-GTP led to a concentration-dependent suppression of [ 3 H]GTP-bound Rac1 but had little effect on [ 3 H]GTP-bound Ras.

    Journal: Journal of Clinical Investigation

    Article Title: CD28-dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes

    doi: 10.1172/JCI200316432

    Figure Lengend Snippet: ( a ) Rac1 activation assay in activated primary T cells. Purified CD4 + T lymphocytes were stimulated with IL-2 and antibodies to CD3 or IL-2 plus antibodies to CD3 and CD28 for 3 days. GTP-bound Rac1 (Rac1-GTP) was analyzed using PAK to determine Rac1 activation. CD28 costimulation led to induction of Rac1 activation in primary T cells. ( b ) Azathioprine and 6-MP suppress Rac1 activation. Purified CD4 + T lymphocytes were stimulated in the presence or absence of azathioprine or 6-MP for 3 days, as indicated. GTP-bound Rac1 was analyzed using PAK to determine Rac1 activation. Azathioprine treatment led to a reduction of CD28-dependent Rac1 activation. One representative experiment out of five is shown. ( c ) 6-MP and 6-TG fail to modulate Ras activation. GTP-bound Ras (Ras-GTP) was analyzed using Raf RGD to determine Ras activation. Azathioprine and its metabolites did not affect Ras activation. One representative experiment of three is shown. ( d ) Downregulation of STAT-3 in primary T cells upon treatment with azathioprine or 6-MP. Purified CD4 + ), GTP-bound Rac1 was obtained using PAK, and STAT-3 levels were determined by immunoblotting. ( e ) Competition of GTP binding to Rac1 or Ras by 6-Thio-GTP. Recombinant Rac1 or Ras was incubated with radiolabeled GTP ([ 3 H]GTP) and increasing amounts of chemically synthesized 6-Thio-GTP (0–500 μM). Next, Rac1 was obtained using PAK-1 agarose, followed by analysis of [ 3 H]GTP-bound Rac1 by scintillation counting. Similarly, Ras was obtained using Raf RGD agarose followed by analysis of [ 3 H]GTP-bound Ras by scintillation counting. 6-Thio-GTP led to a concentration-dependent suppression of [ 3 H]GTP-bound Rac1 but had little effect on [ 3 H]GTP-bound Ras.

    Article Snippet: To analyze the capacity of 6-Thio-GTP to bind to Rac1 or Ras, chemically synthesized 6-Thio-GTP (obtained from Jena Bioscience, Jena, Germany) was used.

    Techniques: Activation Assay, Purification, Binding Assay, Recombinant, Incubation, Synthesized, Concentration Assay

    Vav is required for TCR-driven TEM. A. TEM assays of EM CD4 T cells treated with vehicle and 5 μM 6-Thio GTP for 44 hours. Graphs show %TEM of cells lacking the Vβ2TCR (VB2−, which do not interact with TSST-1 and transmigrate in

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: T cell receptor-driven transendothelial migration of human effector memory CD4 T cells involves Vav, Rac and Myosin IIA

    doi: 10.4049/jimmunol.1201817

    Figure Lengend Snippet: Vav is required for TCR-driven TEM. A. TEM assays of EM CD4 T cells treated with vehicle and 5 μM 6-Thio GTP for 44 hours. Graphs show %TEM of cells lacking the Vβ2TCR (VB2−, which do not interact with TSST-1 and transmigrate in

    Article Snippet: Where indicated, EM CD4 T cells were treated with piceatannol (Calbiochem) and Syk inhibitor II (Calbiochem) to inhibit ZAP-70, with, blebbistatin (Sigma) to inhibit myosin IIA, ML-7 (Calbiochem) to inhibit myosin light chain kinase (MLCK), EHop-016 (Calbiochem) to inhibit Rac and Cdc42, 6-Thio-GTP (Jena Bioscience) to inhibit Vav, or vehicle control (DMSO) and then washed prior to flow TEM assays.

    Techniques: Transmission Electron Microscopy