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. 2010 Jan 12;33(4):861–868. doi: 10.2337/dc09-1799

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© 2010 by the American Diabetes Association.

Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details.

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A: Endotoxin concentration in control (n = 23) and type 2 diabetic (n = 23) subjects was measured using Limulus Ameobocyte lysate assay as described in research design and methods. Values are expressed as EU/ml. *P < 0.05 vs. control. C, control; T2DM, type 2 diabetes. B: HMGB1 concentration in control (n = 23) and type 2 diabetic (n = 23) subjects was measured using ELISA as described in research design and methods. Values are expressed as ng/ml. *P < 0.0001 vs. control. C: Hyaluronan concentration in control (n = 23) and type 2 diabetic (n = 23) subjects was measured using ELISA as described in research design and methods. Values are expressed as ng/ml. *P < 0.0001 vs. control. D: HSP60 concentration in control (n = 23) and type 2 diabetes (n = 23) subjects was measured using ELISA as described in research design and methods. Values are expressed as ng/ml. *P < 0.0001 vs. control. E: HSP70 concentration in control (n = 23) and type 2 diabetic (n = 23) subjects was measured using ELISA as described in research design and methods. Values are expressed as ng/ml. *P < 0.0001 vs. control. F: Association of TLR2 with its ligands and TLR6. Representative Western blot showing enhanced expression of TLR6, HMGB1, hyaluronan, and HSP60 in basal control and type 2 diabetes monocyte cell lysates immunoprecipitated with TLR2 antibody as detailed in research design and methods. β-Actin was used as a loading control. Each assay is repeated four times. IP, immunoprecipitation. G: Association of TLR4 with its ligands. Representative Western blot showing enhanced expression of HMGB1, hyaluronan, and HSP60 in basal control and type 2 diabetes monocyte cell lysates immunoprecipitated with TLR4 antibody as detailed in research design and methods. β-Actin was used as a loading control. Each assay is repeated four times. H: Monocyte TLR signaling in the basal state. TLR downstream signaling proteins MyD88, pIRAK-1, Trif, IRF-3, TECAM-2, and NF-κB p65 were performed using specific antibodies to the respective (phospho) proteins, as described in research design and methods using β-actin as a loading and internal control for MyD88, Trif, IRF-3, TECAM-2, and NF-κB p65 and IRAK for pIRAK-1. Each blot is repeated four times with pooled monocytes from three subjects. Densitometric ratios corroborate the data. *P < 0.05 vs. control. I: The DNA binding activity of nuclear NF-κB p65 in control (n = 23) and type 2 diabetes (n = 23) monocytes was assessed by ELISA as detailed in research design and methods in the basal state. Values are normalized to milligram nuclear protein and expressed as mean ± SD. *P < 0.05 vs. control. J: Serum concentration of cytokines/chemokines in study subjects were measured using Multiplex assays as described in research design and methods. Values are expressed as picograms per milliliter. *P < 0.0001 vs. control. K: Serum concentration of cytokines/chemokines in study subjects were measured using Multiplex assays as described in research design and methods. Values are expressed as picograms per milliliter. *P < 0.0001 vs. control.

A: Endotoxin concentration in control (n = 23) and type 2 diabetic (n = 23) subjects was measured using Limulus Ameobocyte lysate assay as described in research design and methods. Values are expressed as EU/ml. *P < 0.05 vs. control. C, control; T2DM, type 2 diabetes. B: HMGB1 concentration in control (n = 23) and type 2 diabetic (n = 23) subjects was measured using ELISA as described in research design and methods. Values are expressed as ng/ml. *P < 0.0001 vs. control. C: Hyaluronan concentration in control (n = 23) and type 2 diabetic (n = 23) subjects was measured using ELISA as described in research design and methods. Values are expressed as ng/ml. *P < 0.0001 vs. control. D: HSP60 concentration in control (n = 23) and type 2 diabetes (n = 23) subjects was measured using ELISA as described in research design and methods. Values are expressed as ng/ml. *P < 0.0001 vs. control. E: HSP70 concentration in control (n = 23) and type 2 diabetic (n = 23) subjects was measured using ELISA as described in research design and methods. Values are expressed as ng/ml. *P < 0.0001 vs. control. F: Association of TLR2 with its ligands and TLR6. Representative Western blot showing enhanced expression of TLR6, HMGB1, hyaluronan, and HSP60 in basal control and type 2 diabetes monocyte cell lysates immunoprecipitated with TLR2 antibody as detailed in research design and methods. β-Actin was used as a loading control. Each assay is repeated four times. IP, immunoprecipitation. G: Association of TLR4 with its ligands. Representative Western blot showing enhanced expression of HMGB1, hyaluronan, and HSP60 in basal control and type 2 diabetes monocyte cell lysates immunoprecipitated with TLR4 antibody as detailed in research design and methods. β-Actin was used as a loading control. Each assay is repeated four times. H: Monocyte TLR signaling in the basal state. TLR downstream signaling proteins MyD88, pIRAK-1, Trif, IRF-3, TECAM-2, and NF-κB p65 were performed using specific antibodies to the respective (phospho) proteins, as described in research design and methods using β-actin as a loading and internal control for MyD88, Trif, IRF-3, TECAM-2, and NF-κB p65 and IRAK for pIRAK-1. Each blot is repeated four times with pooled monocytes from three subjects. Densitometric ratios corroborate the data. *P < 0.05 vs. control. I: The DNA binding activity of nuclear NF-κB p65 in control (n = 23) and type 2 diabetes (n = 23) monocytes was assessed by ELISA as detailed in research design and methods in the basal state. Values are normalized to milligram nuclear protein and expressed as mean ± SD. *P < 0.05 vs. control. J: Serum concentration of cytokines/chemokines in study subjects were measured using Multiplex assays as described in research design and methods. Values are expressed as picograms per milliliter. *P < 0.0001 vs. control. K: Serum concentration of cytokines/chemokines in study subjects were measured using Multiplex assays as described in research design and methods. Values are expressed as picograms per milliliter. *P < 0.0001 vs. control.