rabbit polyclonal irak1  (Cell Signaling Technology Inc)

Journal: Journal of Thrombosis and Haemostasis

Article Title: Effect of heparanase inhibitor on tissue factor overexpression in platelets and endothelial cells induced by anti‐β2‐GPI antibodies

doi: 10.1111/jth.15417

Figure Lengend Snippet: Heparanase inhibitor RDS3337 reduces interleukin‐1 receptor‐associated kinase 1 (IRAK) phosphorylation and nuclear factor kappa B (NF‐κB) activation triggered by anti‐β2‐glycoprotein I (β2‐GPI) antibodies in endothelial cells. Human umbilical vein endothelial cells were treated for 45 min with affinity‐purified anti‐β2‐GPI antibodies (200 μg/ml) and, as controls, with lipopolysaccharide (LPS; 100 ng/ml) or control human IgG (200 μg/ml). Alternatively, cells were pretreated with heparanase inhibitor RDS3337 (320 nM) for 1 h. After treatments cells were analyzed by western blot for IRAK phosphorylation and NF‐κB‐p65 activation. A, Phosphorylated levels of IRAK1 were evaluated in whole cell extracts using rabbit anti‐phospho‐IRAK1 Ab. The membrane was stripped and reprobed with polyclonal anti‐IRAK1 Ab. For loading control was used anti‐β‐actin mAb. Densitometric phospho‐IRAK1/Total IRAK1 ratios are shown in the right panel. Results represent the mean ± SD from six independent experiments. Statistical analysis indicates: **** P < .0001 versus untreated; §§§§ P < .0001 versus RDS3337 + LPS; °°°° P < .0001 versus RDS3337 + anti‐β2‐GPI antibodies. B, Nuclear cell extracts were analyzed to verify phospho‐NF‐κB‐p65 expression using rabbit anti‐phospho‐NF‐κB‐p65 Ab. As a control, for loading and purity of preparation, membrane was stripped and reprobed with polyclonal anti‐HISTONE H1 Ab. Densitometric phospho‐NF‐κB‐p65/HISTONE H1 ratios are shown in the right panel. Results represent the mean ± SD from six independent experiments. Statistical analysis indicates: **** P < .0001 versus untreated; §§§§ P < .0001 versus RDS3337 + LPS; °°°° P < .0001 versus RDS3337 + anti‐β2‐GPI antibodies

Article Snippet: Proteins were electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (Bio‐Rad Laboratories) and then, after blocking with Tris‐buffered saline Tween 20 (TBS‐T) 3% BSA, incubated with polyclonal rabbit anti‐phospho‐IRAK1 (Cell Signaling, Inc.) or polyclonal rabbit anti‐phospho‐NF‐κB‐p65 antibodies (Cell Signaling, Inc.).

Techniques: Phospho-proteomics, Activation Assay, Affinity Purification, Control, Western Blot, Membrane, Expressing

Journal: Journal of Thrombosis and Haemostasis

Article Title: Effect of heparanase inhibitor on tissue factor overexpression in platelets and endothelial cells induced by anti‐β2‐GPI antibodies

doi: 10.1111/jth.15417

Figure Lengend Snippet: Heparanase inhibitor RDS3337 decreases interleukin‐1 receptor‐associated kinase 1 (IRAK) phosphorylation and nuclear factor kappa B (NF‐κB) activation triggered by anti‐β2‐glycoprotein I (β2‐GPI) antibodies in platelets. Human platelets from healthy donors were treated for 10 min with affinity‐purified anti‐β2‐GPI antibodies (200 μg/ml) and, as controls, with lipopolysaccharide (LPS; 100 ng/ml) or control human IgG (200 μg/ml). Alternatively, cells were pretreated with heparanase inhibitor RDS3337 (320 nM) for 1 h. After treatments cells were analyzed by western blot for IRAK phosphorylation and NF‐κB‐p65 activation. A, Whole platelet extracts were used to determine the levels of phospho‐IRAK1 expression using rabbit anti‐phospho‐IRAK1 Ab. The membrane was stripped and reprobed with polyclonal anti‐IRAK1 Ab. For loading control was used anti‐β‐actin mAb. Densitometric phospho‐IRAK1/Total IRAK1 ratios are shown in the right panel. Results represent the mean ± standard deviation (SD) from six independent experiments. Statistical analysis indicates: **** P < .0001 versus untreated; §§§§ P < .0001 versus RDS3337 + LPS; °°°° P < .0001 versus RDS3337 + anti‐β2‐GPI antibodies. B, Phosphorylated levels of NF‐κB‐p65 were evaluated in whole platelet extracts using anti‐phospho‐NF‐κB‐p65 Ab. The membrane was stripped and reprobed with polyclonal anti‐NF‐κB‐p65 Ab. For loading control was used anti‐β‐actin mAb. Densitometric phospho‐NF‐κB‐p65/NF‐κB‐p65 ratios are shown in the right panel. Results represent the mean ± SD from six independent experiments. Statistical analysis indicates: **** P < .0001 versus untreated; §§§§ P < .0001 versus RDS3337 + LPS; °°°° P < .0001 versus RDS3337 + anti‐β2‐GPI antibodies

Article Snippet: Proteins were electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (Bio‐Rad Laboratories) and then, after blocking with Tris‐buffered saline Tween 20 (TBS‐T) 3% BSA, incubated with polyclonal rabbit anti‐phospho‐IRAK1 (Cell Signaling, Inc.) or polyclonal rabbit anti‐phospho‐NF‐κB‐p65 antibodies (Cell Signaling, Inc.).

Techniques: Phospho-proteomics, Activation Assay, Affinity Purification, Control, Western Blot, Expressing, Membrane, Standard Deviation

Journal: Scientific Reports

Article Title: Extracellular vesicles from T cells overexpress miR-146b-5p in HIV-1 infection and repress endothelial activation

doi: 10.1038/s41598-019-44743-w

Figure Lengend Snippet: miR-146b-EVs repress endothelial activation in vitro via the downregulation of IRAK1 and TRAF6 expression. ( A ) HUVEC were incubated with miR-146b-EVs or Neg-EVs at 37 °C for 48 hours prior to the addition of TNFα or vehicle for 4 hours. ( A ) Western blot analysis of IRAK1, TRAF6, ICAM-1 and VCAM-1 protein expression in HUVEC. Blots come from different parts of the same gel as shown by delineation. After the quantification of protein expression, data were normalized to actin expression and presented relative to the data in unstimulated HUVEC incubated with Neg-EVs. Mean +/− SEM, * P < 0.05, n = 3 to 4. ( B , C ) HUVEC were incubated with miR-146b-EVs or Neg-EVs at 37 °C for 24 hours prior to the addition of TNFα or vehicle for 4 hours. Quantitative RT-PCR analysis of ICAM-1 , VCAM-1 ( B ), IL-6 and IL-8 ( C ) mRNA expression in HUVEC. Data are normalized to GAPDH expression and presented relative to unstimulated HUVEC incubated with Neg-EVs. Mean +/− SEM, * P < 0.05, n = 3.

Article Snippet: The following antibodies were used: rabbit anti-human polyclonal IRAK1 (4504, 1/1,000, Cell Signaling Technology), rabbit polyclonal TRAF6 (8028, 1/1,000, Cell Signaling Technology), rabbit polyclonal ICAM-1 (4915, 1/1,000, Cell Signaling Technology), rabbit polyclonal VCAM-1 (sc8304, 1/200, Santa Cruz Biotechnology), rabbit polyclonal β-actin (8457, 1/3,000, Cell Signaling Technology).

Techniques: Activation Assay, In Vitro, Expressing, Incubation, Western Blot, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Extracellular vesicles from T cells overexpress miR-146b-5p in HIV-1 infection and repress endothelial activation

doi: 10.1038/s41598-019-44743-w

Figure Lengend Snippet: miR-146b-EVs transfer miR-146b-5p mimics to lungs in vivo and repress endothelial activation in vivo . ( A ) Flow cytometric analyses of lungs, heart and aorta from mice injected with Syto control (gray curve) or with Syto-EVs (green curve) after 30 minutes or 20 hours. Data are representative of 3 different experiments. ( B) Mice were injected with miR-146b-EVs or Neg-EVs for 24 hours. miR-146-5p expression level in lungs, heart and aorta was determined by quantitative RT-PCR. Data are normalized to U6 snRNA expression and compared to the values in each organ from mice injected with Neg-EVs. Mean +/− SEM, * P < 0.05, n = 4 to 5. ( C , D ) Mice were injected with miR-146b-EVs or Neg-EVs 24 hours prior to the injection of vehicle or TNFα for 4 hours. Data are presented relative to values in vehicle injected-mice treated with Neg-EVs. Mean +/− SEM, * P < 0.05, n = 4 to 5. ( C ) Quantitative RT-PCR analysis of IRAK1 , TRAF6 , ICAM-1 and VCAM-1 mRNA expression in lungs. Data are normalized to GAPDH expression. ( D ), Representative micrographs of lungs with VCAM-1 staining, and quantitative analysis of VCAM-1 staining.

Article Snippet: The following antibodies were used: rabbit anti-human polyclonal IRAK1 (4504, 1/1,000, Cell Signaling Technology), rabbit polyclonal TRAF6 (8028, 1/1,000, Cell Signaling Technology), rabbit polyclonal ICAM-1 (4915, 1/1,000, Cell Signaling Technology), rabbit polyclonal VCAM-1 (sc8304, 1/200, Santa Cruz Biotechnology), rabbit polyclonal β-actin (8457, 1/3,000, Cell Signaling Technology).

Techniques: In Vivo, Activation Assay, Injection, Control, Expressing, Quantitative RT-PCR, Staining

Journal: Scientific Reports

Article Title: Extracellular vesicles from T cells overexpress miR-146b-5p in HIV-1 infection and repress endothelial activation

doi: 10.1038/s41598-019-44743-w

Figure Lengend Snippet: Pathophysiological hypothesis. Proposed model of how CD4 T cell-derived EVs act in vascular repair by counteracting endothelial activation in HIV-1 infection. Upon HIV-1 infection, the miRNA contents of CD4 T cells and CD4 T cell-derived EVs are altered, especially miR-146b-5p, which is upregulated. miR-146b-5p is incorporated into CD4 T cell-derived EVs and could be protected from RNase degradation in the bloodstream. CD4 T cell-derived EVs transfer miR-146b-5p to endothelial cells in a phosphatidylserine (PS)-dependent manner and lead to a decreased expression of nuclear factor-κB (NF-κB)-responsive molecules, including ICAM-1 and VCAM-1, through the downregulation of IRAK1 and TRAF6.

Article Snippet: The following antibodies were used: rabbit anti-human polyclonal IRAK1 (4504, 1/1,000, Cell Signaling Technology), rabbit polyclonal TRAF6 (8028, 1/1,000, Cell Signaling Technology), rabbit polyclonal ICAM-1 (4915, 1/1,000, Cell Signaling Technology), rabbit polyclonal VCAM-1 (sc8304, 1/200, Santa Cruz Biotechnology), rabbit polyclonal β-actin (8457, 1/3,000, Cell Signaling Technology).

Techniques: Derivative Assay, Activation Assay, Infection, Expressing