Journal: Mediators of Inflammation

Article Title: Mycobacterium tuberculosis Requires Cholesterol Oxidase to Disrupt TLR2 Signalling in Human Macrophages

doi: 10.1155/2019/2373791

Figure Lengend Snippet: IRAK4 and p-IRAK4 protein levels in Mtb-infected macrophages. The macrophages were infected with (a) wild-type, (b) Δ choD , or (c) Δ choD-choD strains for 0.5 to 24 hours. The IRAK4 protein level was assessed using the immunoblot-ECL method. Representative immunoblots of the total and phosphorylated IRAK4 protein level are shown. The bands were quantified by densitometric analysis. The data are presented as the optical density intensity of the area under each band′s peak (ODI) ± SEM from 5 independent experiments. ∗ Macrophages infected with the wild-type or Δ choD-choD vs. noninfected macrophages, p ≤ 0.02 (Wilcoxon's signed rank test).

Article Snippet: The membranes were blocked in SuperBlock Blocking Buffer and then probed with the following four specific primary antibodies: (1) rabbit anti-IRAK4 polyclonal antibody (1 : 1000), (2) rabbit anti-phospho-IRAK4 (Thr345/Ser346) monoclonal antibody (1 : 1000), (3) rabbit anti-beta-actin monoclonal antibody (1 : 4000) (Cell Signaling Technology, Danvers, USA), or (4) rabbit anti-TRAF6 polyclonal antibody (1 : 1000) (Proteintech Group, Rosemont, USA).

Techniques: Infection, Western Blot

Journal: Mediators of Inflammation

Article Title: Mycobacterium tuberculosis Requires Cholesterol Oxidase to Disrupt TLR2 Signalling in Human Macrophages

doi: 10.1155/2019/2373791

Figure Lengend Snippet: IRAK4 and TRAF6 protein levels in Mtb-infected macrophages with blocked TLR2 expression. The macrophages were incubated with an anti-human TLR2 monoclonal antibody (a) or isotype control (b) and then infected with the wild-type, Δ choD , or Δ choD-choD strains for 24 hours. Next, the cells were lysed, and the protein level was assessed using the immunoblot-ECL method. Representative immunoblots of the IRAK4 and TRAF6 protein levels are shown. The bands were quantified by densitometric analysis. The data are presented as the optical density intensity of the area under each band′s peak (ODI) ± SEM from 3 independent experiments.

Article Snippet: The membranes were blocked in SuperBlock Blocking Buffer and then probed with the following four specific primary antibodies: (1) rabbit anti-IRAK4 polyclonal antibody (1 : 1000), (2) rabbit anti-phospho-IRAK4 (Thr345/Ser346) monoclonal antibody (1 : 1000), (3) rabbit anti-beta-actin monoclonal antibody (1 : 4000) (Cell Signaling Technology, Danvers, USA), or (4) rabbit anti-TRAF6 polyclonal antibody (1 : 1000) (Proteintech Group, Rosemont, USA).

Techniques: Infection, Expressing, Incubation, Control, Western Blot

Journal: Mediators of Inflammation

Article Title: Mycobacterium tuberculosis Requires Cholesterol Oxidase to Disrupt TLR2 Signalling in Human Macrophages

doi: 10.1155/2019/2373791

Figure Lengend Snippet: IRAK4 and TRAF6 protein levels in macrophages treated with PMA, IL-1 β , or LTA. The macrophages were cultured for 24 hours in the presence of PMA, lipoteichoic acid (LTA), or human recombinant interleukin 1 β (hrIL-1 β ). Next, the cells were lysed, and the IRAK4, p-IRAK4, and TRAF6 protein levels were assessed using the immunoblot-ECL method. Representative immunoblots of the IRAK4 and TRAF6 protein levels are shown. The bands were quantified by densitometric analysis. The data are presented as the optical density intensity of the area under each band's peak (ODI)±SEM from 2 independent experiments.

Article Snippet: The membranes were blocked in SuperBlock Blocking Buffer and then probed with the following four specific primary antibodies: (1) rabbit anti-IRAK4 polyclonal antibody (1 : 1000), (2) rabbit anti-phospho-IRAK4 (Thr345/Ser346) monoclonal antibody (1 : 1000), (3) rabbit anti-beta-actin monoclonal antibody (1 : 4000) (Cell Signaling Technology, Danvers, USA), or (4) rabbit anti-TRAF6 polyclonal antibody (1 : 1000) (Proteintech Group, Rosemont, USA).

Techniques: Cell Culture, Recombinant, Western Blot

Journal: Mediators of Inflammation

Article Title: Mycobacterium tuberculosis Requires Cholesterol Oxidase to Disrupt TLR2 Signalling in Human Macrophages

doi: 10.1155/2019/2373791

Figure Lengend Snippet: The IRAK4 and TRAF6 mRNA levels in Mtb-infected macrophages. The macrophages were infected with the wild-type, Δ choD , or Δ choD-choD strains for 0.5 to 24 hours, and the total RNA was isolated. The IRAK4 and TRAF6 mRNA level was measured using qRT-PCR. The data are presented as the mean relative quantification (RQ) ± SEM from 5 independent experiments. The solid line marks the level of gene expression in control cells (noninfected macrophages). RQ represents the fold change in the gene expression in the infected macrophages compared to the noninfected macrophages, calculated using the ABI 7900HT RQ Manager Software v1.2 and DataAssist Software v3.01 (Thermo Fisher Scientific). ∗ Macrophages infected with wild-type, Δ choD , or Δ choD-choD Mtb vs. noninfected macrophages, p ≤ 0.03; # Macrophages infected with Δ choD Mtb vs. macrophages infected with wild-type Mtb, p ≤ 0.04 ( t -test).

Article Snippet: The membranes were blocked in SuperBlock Blocking Buffer and then probed with the following four specific primary antibodies: (1) rabbit anti-IRAK4 polyclonal antibody (1 : 1000), (2) rabbit anti-phospho-IRAK4 (Thr345/Ser346) monoclonal antibody (1 : 1000), (3) rabbit anti-beta-actin monoclonal antibody (1 : 4000) (Cell Signaling Technology, Danvers, USA), or (4) rabbit anti-TRAF6 polyclonal antibody (1 : 1000) (Proteintech Group, Rosemont, USA).

Techniques: Infection, Isolation, Quantitative RT-PCR, Quantitative Proteomics, Gene Expression, Control, Software

Journal: Mediators of Inflammation

Article Title: Mycobacterium tuberculosis Requires Cholesterol Oxidase to Disrupt TLR2 Signalling in Human Macrophages

doi: 10.1155/2019/2373791

Figure Lengend Snippet: The effect of TBChoD recombinant protein on TLR2, TRAF6, IRAK4, and p-IRAK4 levels in macrophages. (a) The macrophages were treated with a range concentration of TBChoD recombinant protein (1-25 μ g/ml) for 24 hours. The cells were then stained with the PE-conjugated anti-TLR2 mAb or a specific isotype control for 30 minutes. The TLR2 expression on the macrophages was analysed using flow cytometry. The graphs show the mean values of the mean fluorescence intensity (MFI) ± SEM from 5 independent experiments. (b, c) The macrophages were treated with 10 μ g/ml of TBChoD recombinant protein for 1, 2, or 4 hours. The TRAF6, IRAK4, and p-IRAK4 protein levels were assessed using the immunoblot-ECL method. Representative immunoblots of abovementioned proteins are shown. The bands were quantified by densitometric analysis. The data are presented as the optical density intensity of the area under each band′s peak (ODI) ± SEM from 3 independent experiments. ∗ Macrophages treated with TBChoD protein vs. nontreated macrophages, p ≤ 0.05 (Wilcoxon's signed rank test).

Article Snippet: The membranes were blocked in SuperBlock Blocking Buffer and then probed with the following four specific primary antibodies: (1) rabbit anti-IRAK4 polyclonal antibody (1 : 1000), (2) rabbit anti-phospho-IRAK4 (Thr345/Ser346) monoclonal antibody (1 : 1000), (3) rabbit anti-beta-actin monoclonal antibody (1 : 4000) (Cell Signaling Technology, Danvers, USA), or (4) rabbit anti-TRAF6 polyclonal antibody (1 : 1000) (Proteintech Group, Rosemont, USA).

Techniques: Recombinant, Concentration Assay, Staining, Control, Expressing, Flow Cytometry, Fluorescence, Western Blot

Journal: Mediators of Inflammation

Article Title: Capsular Polysaccharide is a Main Component of Mycoplasma ovipneumoniae in the Pathogen-Induced Toll-Like Receptor-Mediated Inflammatory Responses in Sheep Airway Epithelial Cells

doi: 10.1155/2017/9891673

Figure Lengend Snippet: Sheep gene specific primers for qRT-PCR.

Article Snippet: Antibodies used in this study included rabbit anti-Toll-like receptor 4, rabbit anti-AP-1, rabbit anti-p-AP-1 (Cell Signaling Technology, Beverly, MA, USA), rabbit anti-MyD88, rabbit anti-IRAK1, rabbit anti-IRAK4, rabbit anti-TRAF6, rabbit anti-TAB1, rabbit anti-TRIF, rabbit anti-TRAF3, rabbit anti-IRF3, rabbit anti-IRF5, rabbit anti- β -actin (Proteintech Group, Chicago, USA), rabbit anti-IRAK2, rabbit anti-TBK1 (ABGENT, San Diego, USA), and rabbit anti-p-NFkB (Signalway Antibody, Maryland, USA).

Techniques: Sequencing

Journal: Mediators of Inflammation

Article Title: Capsular Polysaccharide is a Main Component of Mycoplasma ovipneumoniae in the Pathogen-Induced Toll-Like Receptor-Mediated Inflammatory Responses in Sheep Airway Epithelial Cells

doi: 10.1155/2017/9891673

Figure Lengend Snippet: The impact of CPS on the expression of MyD88-dependent TLR signaling pathway in ALI cultures of sheep bronchial epithelial cells. 4-week old ALI cultures of sheep bronchial epithelial cells were exposed to indicated conditions for 48 h. (a) The expression of several key components of MyD88-dependent signaling cascade, including the MyD88, IRAK1, IRAK2, IRAK4, TRAF6, and TAB1 transcripts were determined by qRT-PCR assays. In comparison with the controls, all tested transcripts showed a significant increase in the cells treated with CPS or M. ovipneumoniae . (b) Immunoblotting assay also showed an evoked expression of all MyD88-dependent signaling-associated proteins in ALI cultures upon CPS/ MO. ovipneumoniae stimulation. (c) Densitometric analysis of western blot showed MyD88, IRAK1, IRAK2, IRAK4, TRAF6, and TAB1 expression over β -actin. Values are mean ± SD for at least three independent experiments performed in triplicate. ∗∗ p < 0.01 versus that of the control. Compared between indicated groups, ▲▲ p < 0.01.

Article Snippet: Antibodies used in this study included rabbit anti-Toll-like receptor 4, rabbit anti-AP-1, rabbit anti-p-AP-1 (Cell Signaling Technology, Beverly, MA, USA), rabbit anti-MyD88, rabbit anti-IRAK1, rabbit anti-IRAK4, rabbit anti-TRAF6, rabbit anti-TAB1, rabbit anti-TRIF, rabbit anti-TRAF3, rabbit anti-IRF3, rabbit anti-IRF5, rabbit anti- β -actin (Proteintech Group, Chicago, USA), rabbit anti-IRAK2, rabbit anti-TBK1 (ABGENT, San Diego, USA), and rabbit anti-p-NFkB (Signalway Antibody, Maryland, USA).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: International immunopharmacology

Article Title: Artemisinin attenuates perinatal inflammation and consequent oxidative stress in oligodendrocyte precursor cells by inhibiting IRAK-4 and IRAK-1.

doi: 10.1016/j.intimp.2024.113117

Figure Lengend Snippet: Fig. 3. Docking analysis and CETSA results showing that IRAK-4 and IRAK-1 are both potential target proteins for ART. PyMOL and AutoDock were used for molecular modelling (Homo sapiens: irak4, PDB ID: 6O94; irak1, PDB ID: 6BFN) (Rattus norvegicus: irak4, UniProt: D4A7K4; irak1, UniProt: B2RYH5), and analysis of properties and binding sites (ART and IRAK-4; ART and IRAK-1) revealed that ART mainly forms hydrogen bonds and binds to sites near phosphorylation sites. b, c, d, e A CETSA was used to assess the binding of ART to its targets over a predefined temperature gradient (gradient: 45.0, 46.8, 49.8, 54.4, 59.9, 64.8, 68.0, and 70.0 ◦C). Western blot analysis of differences in protein expression (IRAK-4, IRAK-1, and β-actin) between the Ctrl and ART (2.5 μM) groups. Quantification of Band intensity. The data are expressed as the mean ± SEM (n = 6/group). *p < 0.05 and **p < 0.01 versus the indicated groups.

Article Snippet: For immunofluorescence, antigen retrieval was performed using Quick Antigen Retrieval Solution for Frozen Sections (P0090, Beyond, Shanghai, China), and the tissues were incubated with 0.3 % Triton X100 for 10 min, blocked and incubated with primary antibodies against phospho-IRAK4 (p-IRAK-4) (Thr345/Ser346) (1:100, DF7567, Affinity, Jiangsu, China), phospho-IRAK1 (p-IRAK-4) (Thr387) (1:100, AF8009, Affinity), NF-kB p65 (1:100, AF5006, Affinity), and Nrf2 (1:100, AF0639, Affinity) for 16 h. The samples were incubated with a goat antirat IgG antibody (1:200, SA00003-11, Proteintech) labelled with FITC, a goat anti-mouse IgG antibody (1:100, SA00009-1, Proteintech) labelled with Cy3, and a goat anti-rabbit IgG antibody (1:200, SA00003-2, Proteintech) labelled with FITC at 37 ◦C in the dark for 1.5 h. For immunohistochemistry, the tissues were incubated with enzyme-conjugated goat anti-mouse immunoglobulin G secondary antibody (SP-900; ZSGBBio, Beijing, China) solution for 15 min at 37 ◦C.

Techniques: Binding Assay, Phospho-proteomics, Western Blot, Expressing

Journal: International immunopharmacology

Article Title: Artemisinin attenuates perinatal inflammation and consequent oxidative stress in oligodendrocyte precursor cells by inhibiting IRAK-4 and IRAK-1.

doi: 10.1016/j.intimp.2024.113117

Figure Lengend Snippet: Fig. 4. LPS-induced activation of the IRAK-4/IRAK-1/NF-κB signalling pathway in OPCs was inhibited by ART. a, b, c, d Western blotting was used to quantify the protein levels of p-IRAK-4, IRAK-4, p-IRAK-4/IRAK-4, p-IRAK-1, IRAK-1, p-IRAK-1/IRAK-1, phospho-NF-kB p65, NF-κB p65, and phospho-NF-kB p65/NF-κB p65 in 4 groups (the Ctrl, ART, LPS, and LPS + ART groups). The data are expressed as the means ± SEMs (n = 6/group) (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001).

Article Snippet: For immunofluorescence, antigen retrieval was performed using Quick Antigen Retrieval Solution for Frozen Sections (P0090, Beyond, Shanghai, China), and the tissues were incubated with 0.3 % Triton X100 for 10 min, blocked and incubated with primary antibodies against phospho-IRAK4 (p-IRAK-4) (Thr345/Ser346) (1:100, DF7567, Affinity, Jiangsu, China), phospho-IRAK1 (p-IRAK-4) (Thr387) (1:100, AF8009, Affinity), NF-kB p65 (1:100, AF5006, Affinity), and Nrf2 (1:100, AF0639, Affinity) for 16 h. The samples were incubated with a goat antirat IgG antibody (1:200, SA00003-11, Proteintech) labelled with FITC, a goat anti-mouse IgG antibody (1:100, SA00009-1, Proteintech) labelled with Cy3, and a goat anti-rabbit IgG antibody (1:200, SA00003-2, Proteintech) labelled with FITC at 37 ◦C in the dark for 1.5 h. For immunohistochemistry, the tissues were incubated with enzyme-conjugated goat anti-mouse immunoglobulin G secondary antibody (SP-900; ZSGBBio, Beijing, China) solution for 15 min at 37 ◦C.

Techniques: Activation Assay, Western Blot

Journal: International immunopharmacology

Article Title: Artemisinin attenuates perinatal inflammation and consequent oxidative stress in oligodendrocyte precursor cells by inhibiting IRAK-4 and IRAK-1.

doi: 10.1016/j.intimp.2024.113117

Figure Lengend Snippet: Fig. 5. ART reduces NF-κB nuclear aggregation after LPS stimulation. a, b, c Immunofluorescence was used to compare the relative fluorescence of p-IRAK-4 (red) and p-IRAK-1 (red) in OPCs across several groups (the Ctrl, ART, LPS, and LPS + ART groups), and the proportion (%) of nuclear NF-κB p65 (red) was determined. DAPI staining is shown in blue. Scale bar = 50 μm. The data are presented as the means ± SEMs (n = 6). ****P < 0.0001 versus the indicated groups.

Article Snippet: For immunofluorescence, antigen retrieval was performed using Quick Antigen Retrieval Solution for Frozen Sections (P0090, Beyond, Shanghai, China), and the tissues were incubated with 0.3 % Triton X100 for 10 min, blocked and incubated with primary antibodies against phospho-IRAK4 (p-IRAK-4) (Thr345/Ser346) (1:100, DF7567, Affinity, Jiangsu, China), phospho-IRAK1 (p-IRAK-4) (Thr387) (1:100, AF8009, Affinity), NF-kB p65 (1:100, AF5006, Affinity), and Nrf2 (1:100, AF0639, Affinity) for 16 h. The samples were incubated with a goat antirat IgG antibody (1:200, SA00003-11, Proteintech) labelled with FITC, a goat anti-mouse IgG antibody (1:100, SA00009-1, Proteintech) labelled with Cy3, and a goat anti-rabbit IgG antibody (1:200, SA00003-2, Proteintech) labelled with FITC at 37 ◦C in the dark for 1.5 h. For immunohistochemistry, the tissues were incubated with enzyme-conjugated goat anti-mouse immunoglobulin G secondary antibody (SP-900; ZSGBBio, Beijing, China) solution for 15 min at 37 ◦C.

Techniques: Immunofluorescence, Fluorescence, Staining

Journal: International immunopharmacology

Article Title: Artemisinin attenuates perinatal inflammation and consequent oxidative stress in oligodendrocyte precursor cells by inhibiting IRAK-4 and IRAK-1.

doi: 10.1016/j.intimp.2024.113117

Figure Lengend Snippet: Fig. 7. The IRAK-4/IRAK-1/NF-κB signalling pathway in OPCs in the white matter is markedly suppressed in the presence of perinatal inflammation following ART treatment. a, b, c, d Western blotting was used to quantify the protein levels of p-IRAK-4, IRAK-4, p-IRAK-4/IRAK-4, p-IRAK-1, IRAK-1, p-IRAK-1/IRAK-1, phospho- NF-kB p65, NF-κB p65, and phospho-NF-kB p65/NF-κB p65 in 4 groups (the Ctrl, ART, LPS, and LPS + ART groups) at P2. The data are expressed as the means ± SEMs (n = 6 rats/group) (*P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001).

Article Snippet: For immunofluorescence, antigen retrieval was performed using Quick Antigen Retrieval Solution for Frozen Sections (P0090, Beyond, Shanghai, China), and the tissues were incubated with 0.3 % Triton X100 for 10 min, blocked and incubated with primary antibodies against phospho-IRAK4 (p-IRAK-4) (Thr345/Ser346) (1:100, DF7567, Affinity, Jiangsu, China), phospho-IRAK1 (p-IRAK-4) (Thr387) (1:100, AF8009, Affinity), NF-kB p65 (1:100, AF5006, Affinity), and Nrf2 (1:100, AF0639, Affinity) for 16 h. The samples were incubated with a goat antirat IgG antibody (1:200, SA00003-11, Proteintech) labelled with FITC, a goat anti-mouse IgG antibody (1:100, SA00009-1, Proteintech) labelled with Cy3, and a goat anti-rabbit IgG antibody (1:200, SA00003-2, Proteintech) labelled with FITC at 37 ◦C in the dark for 1.5 h. For immunohistochemistry, the tissues were incubated with enzyme-conjugated goat anti-mouse immunoglobulin G secondary antibody (SP-900; ZSGBBio, Beijing, China) solution for 15 min at 37 ◦C.

Techniques: Western Blot

Journal: International immunopharmacology

Article Title: Artemisinin attenuates perinatal inflammation and consequent oxidative stress in oligodendrocyte precursor cells by inhibiting IRAK-4 and IRAK-1.

doi: 10.1016/j.intimp.2024.113117

Figure Lengend Snippet: Fig. 8. ART reduces NF-κB nuclear translocation in white matter OPCs. a, b, c Immunofluorescence staining of white matter (P2) was performed to compare the relative fluorescence of p-IRAK-4 (green) and p-IRAK-1 (green) in OPCs (PDGFRα+, red) across several groups (the Ctrl, ART, LPS, and LPS + ART groups), and the proportion (%) of nuclear NF-κB p65 (green) was determined. DAPI staining is shown in blue. Scale bar = 50 μm. The data are presented as the means ± SEMs (n = 6 rats/group). *P < 0.05, ***P < 0.001 and ****P < 0.0001 versus the indicated groups.

Article Snippet: For immunofluorescence, antigen retrieval was performed using Quick Antigen Retrieval Solution for Frozen Sections (P0090, Beyond, Shanghai, China), and the tissues were incubated with 0.3 % Triton X100 for 10 min, blocked and incubated with primary antibodies against phospho-IRAK4 (p-IRAK-4) (Thr345/Ser346) (1:100, DF7567, Affinity, Jiangsu, China), phospho-IRAK1 (p-IRAK-4) (Thr387) (1:100, AF8009, Affinity), NF-kB p65 (1:100, AF5006, Affinity), and Nrf2 (1:100, AF0639, Affinity) for 16 h. The samples were incubated with a goat antirat IgG antibody (1:200, SA00003-11, Proteintech) labelled with FITC, a goat anti-mouse IgG antibody (1:100, SA00009-1, Proteintech) labelled with Cy3, and a goat anti-rabbit IgG antibody (1:200, SA00003-2, Proteintech) labelled with FITC at 37 ◦C in the dark for 1.5 h. For immunohistochemistry, the tissues were incubated with enzyme-conjugated goat anti-mouse immunoglobulin G secondary antibody (SP-900; ZSGBBio, Beijing, China) solution for 15 min at 37 ◦C.

Techniques: Translocation Assay, Immunofluorescence, Staining, Fluorescence