Journal: Acta Pharmacologica Sinica

Article Title: Polydatin ameliorates Staphylococcus aureus -induced mastitis in mice via inhibiting TLR2-mediated activation of the p38 MAPK/NF-κB pathway

doi: 10.1038/aps.2016.123

Figure Lengend Snippet: The effects of PD on the TLR2-mediated activation of the MAPK signaling pathway in tissues. The expression of MyD88, IRAK1, IRAK4, TRAF6, TAK1, MKK3/6 and MAPK protein in tissues was determined using a Western blot. β-Actin was used as a control. CG is the control group; SG is the S aureus group; DEX is the dexamethasone group; and H, M and L are the PD administration groups, with doses of 45, 30 and 15 mg/kg per animal, respectively, and 100, 50 and 25 μg/mL in the cells, respectively. The data are presented as the mean±SEM. *P<0.05 vs SG. #P<0.05 vs CG.

Article Snippet: Primary antibodies against TLR2, MyD88, IRAK1, IRAK4, TRAF6, TAK1, p-TAK1, MKK3/6, p-MKK3/6, MAPKs, NF-κB, IκBα, p-IκBα and β-actin and the HRP-conjugated goat anti-rabbit antibody were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Activation Assay, Expressing, Western Blot, Control

Journal: Acta Pharmacologica Sinica

Article Title: Polydatin ameliorates Staphylococcus aureus -induced mastitis in mice via inhibiting TLR2-mediated activation of the p38 MAPK/NF-κB pathway

doi: 10.1038/aps.2016.123

Figure Lengend Snippet: The effects of PD on the TLR2-mediated activation of the MAPK signaling pathway in mMECs. The expression of MyD88, IRAK1, IRAK4, TRAF6, TAK1, MKK3/6 and MAPK protein in mMECs was determined using Western blotting. β-Actin was used as a control. CG is the control group; SG is the S aureus group; DEX is the dexamethasone group; and H, M and L are the PD administration groups, with doses of 45, 30 and 15 mg/kg per animal, respectively, and 100, 50 and 25 μg/mL in the cells, respectively. The data are presented as the mean±SEM. *P<0.05 vs SG. #P<0.05 vs CG.

Article Snippet: Primary antibodies against TLR2, MyD88, IRAK1, IRAK4, TRAF6, TAK1, p-TAK1, MKK3/6, p-MKK3/6, MAPKs, NF-κB, IκBα, p-IκBα and β-actin and the HRP-conjugated goat anti-rabbit antibody were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Activation Assay, Expressing, Western Blot, Control

Journal: Journal of Ayurveda and Integrative Medicine

Article Title: Exploring the anti-inflammatory effects of acupuncture and Chinese herbal medicine targeting the miRNA-146a/IRAK1/JNK1 pathway in an environmental dry eye rat model

doi: 10.1016/j.jaim.2024.100998

Figure Lengend Snippet: Protein Expression of IRAK1 and p -JNK1 in corneal tissue of rats. Control: blank control group. Model : model group. SH : 0.3% sodium hyaluronate group. Acup : Acupuncture group. YQCM : classic decoction of “Yiqi Congming Tang”. Note: Compare with Control group ∗ P ＜0.05，∗∗ P ＜0.01; Compared with Model group, # P ＜0.05， ## P ＜0.01; Compare with SH group, ▲ P ＜0.05， ▲▲ P ＜0.01; Compare with Acup group, △ P ＜0.05， △△ P ＜0.01; Compare with YQCM group, ◇ P ＜0.05， ◇◇ P ＜0.01.

Article Snippet: Antibodies : The following antibodies were used: Anti-MMP-9 antibody (abcam, ab76003); Anti-TNF-α antibody (Affinity, AF7014); Anti-IL-1β antibody (Affinity, AF103); Anti-Caspase-3 antibody (bioss, bs-0081R); Anti-IL-6 antibody (bioss, BS-0782R); Anti–IFN–γ antibody (bioss, bs-0480R); Anti-JNK1 antibody (proteintech, 66210-1-lg); Anti-IRAK1 antibody (proteintech, 10478-2-AP); Anti-JNK1 (phospho T183) antibody (abcam, ab47337); Anti-β-actin antibody (abcam, ab115777).

Techniques: Expressing, Control

Journal: Journal of Innate Immunity

Article Title: Tollip Inhibits ST2 Signaling in Airway Epithelial Cells Exposed to Type 2 Cytokines and Rhinovirus

doi: 10.1159/000497072

Figure Lengend Snippet: IRAK1 activation in Tollip-deficient (Tollip-shRNA) and Tollip-sufficient (CTL-shRNA) primary human tracheobronchial epithelial cells. a Representative Western blot showing p-IRAK1 and total IRAK1 expression levels. RV, rhinovirus. b Densitometric analysis of p-IRAK1 expression that was normalized to total IRAK1 expression. Results are expressed as fold change relative to unstimulated control (Med). Data are means ± SEM of n = 4 donors.

Article Snippet: To establish a stable IRAK1-deficient primary HTBE cell line, we transduced primary HTBE cells with shRNA targeting human IRAK1 (GeneCopoeia), using lentivirus as described above for Tollip knockdown.

Techniques: Activation Assay, shRNA, Western Blot, Expressing, Control

Journal: Journal of Innate Immunity

Article Title: Tollip Inhibits ST2 Signaling in Airway Epithelial Cells Exposed to Type 2 Cytokines and Rhinovirus

doi: 10.1159/000497072

Figure Lengend Snippet: IRAK1 requirement for IL-8 production by primary human tracheobronchial epithelial (HTBE) cells. a Western blot confirming IRAK1-shRNA knockdown in primary HTBE cells. b IL-8 levels measured in IRAK1-deficient (IRAK1-shRNA) and IRAK1-sufficient (CTL-shRNA) HTBE cell culture supernatants at baseline (Med) and after infection with rhinovirus (RV) 16 in the presence of IL-13 + IL-33 (RV + IL-13 + 33). Data are means ± SEM of n = 4 replicates for each condition. * p < 0.001, compared to CTL-shRNA for similar treatment.

Article Snippet: To establish a stable IRAK1-deficient primary HTBE cell line, we transduced primary HTBE cells with shRNA targeting human IRAK1 (GeneCopoeia), using lentivirus as described above for Tollip knockdown.

Techniques: Western Blot, shRNA, Knockdown, Cell Culture, Infection

Journal: Journal of Innate Immunity

Article Title: Tollip Inhibits ST2 Signaling in Airway Epithelial Cells Exposed to Type 2 Cytokines and Rhinovirus

doi: 10.1159/000497072

Figure Lengend Snippet: Effect of Tollip gene knockout and IRAK1 inhibitor on IL-8 production by primary human tracheobronchial epithelial (HTBE) cells. a Tollip and IRAK1 protein expression in Tollip knockout (KO) (Tollip-CRISPR) and control (CTL-CRISPR) HTBE cells. b IL-8 production at baseline (Med) and after rhinovirus (RV) infection in the presence of IL-13 + IL-33 (RV + IL-13 + 33). c Effect of an IRAK1 inhibitor on IL-8 production by Tollip KO HTBE cells after infection with RV16 in the presence of IL-13 + IL-33 (RV + IL-13 + 33). IRAK1 inhibitor (5 µM) was added to the culture 2 h before RV infection. IL-8 was measured 24 h after the infection. Data are means ± SEM of n = 4 replicates for each condition. * p < 0.01, compared to the respective baseline control (Med).

Article Snippet: To establish a stable IRAK1-deficient primary HTBE cell line, we transduced primary HTBE cells with shRNA targeting human IRAK1 (GeneCopoeia), using lentivirus as described above for Tollip knockdown.

Techniques: Gene Knockout, Expressing, Knock-Out, CRISPR, Control, Infection

Journal: Journal of Innate Immunity

Article Title: Tollip Inhibits ST2 Signaling in Airway Epithelial Cells Exposed to Type 2 Cytokines and Rhinovirus

doi: 10.1159/000497072

Figure Lengend Snippet: Proposed mechanism of Tollip-regulated IL-8 production in human airway epithelial cells. During rhinovirus (RV) infection in the presence of the pro-asthma cytokines IL-13 and IL-33, Tollip promotes soluble ST2 (sST2) production but limits IRAK1 activation, thereby restricting excessive IL-8 production and the development of detrimental inflammation. Targeting ST2 signaling with exogenous sST2 and inhibiting IRAK1 may be potential avenues for the therapeutic control of excessive airway inflammation during RV-mediated asthma exacerbations.

Article Snippet: To establish a stable IRAK1-deficient primary HTBE cell line, we transduced primary HTBE cells with shRNA targeting human IRAK1 (GeneCopoeia), using lentivirus as described above for Tollip knockdown.

Techniques: Infection, Activation Assay, Control

Journal: Journal of Virology

Article Title: Interleukin-1 Receptor-Associated Kinase (IRAK) Signaling in Kaposi Sarcoma-Associated Herpesvirus-Induced Primary Effusion Lymphoma

doi: 10.1128/JVI.02123-19

Figure Lengend Snippet: IRAK1 is not required for PEL survival. (A) IRAK1 Western blot of BCBL-1Cas9 cell lines showing complete knockout; loading control is β-actin. (B) Growth curves for BCBL-1Cas9 ΔIRAK1 clones obtained via trypan blue cell counting. Two ΔIRAK1 clones and an empty vector control were used in this experiment. (C) Representative images from colony formation assays of ΔIRAK1 BCBL-1Cas9 cells imaged at ×10 magnification. Cells were plated at a low cell density in 1% methylcellulose medium and grown for 3 weeks. (D) Quantification of colony formation in BCBL-1Cas9 ΔIRAK1 stable cell lines. Colony counts were obtained using ImageJ, and the square root of the number of colonies was plotted; n = 15. (E) Flanking cut-site PCR analysis using PerkinElmer LabChip GX-Touch. Primers were designed flanking the cut site. Image analysis revealed changes in band size of the KO versus that of WT cells.

Article Snippet: We obtained three IRAK1 expression plasmids from OriGene, namely, pDD1951 (RC221544, PEL phenotype full-length IRAK1), pDD1952 (RC224107, IRAK1 isoform B), pDD1953 (RC204869, IRAK1 isoform C), and the empty vector control pDD1957 (EV, pCMV6-entry).

Techniques: Western Blot, Knock-Out, Clone Assay, Cell Counting, Plasmid Preparation, Stable Transfection

Journal: Journal of Virology

Article Title: Interleukin-1 Receptor-Associated Kinase (IRAK) Signaling in Kaposi Sarcoma-Associated Herpesvirus-Induced Primary Effusion Lymphoma

doi: 10.1128/JVI.02123-19

Figure Lengend Snippet: MYD88, IRAK1, and IRAK4 are dispensable in BC-1 cells. (A) MYD88 Western blot of BC-1Cas9 cell lines showing complete knockout; loading control is β-actin. (B) IRAK1 Western blot. (C) IRAK4 western blot. (D) Growth curves for BC-1Cas9 ΔMYD88 clones obtained via trypan blue cell counting. Two ΔMYD88 clones and an empty-vector WT control were used in this experiment. (E) Growth curves for BC-1Cas9 ΔIRAK1 clones. (F) Growth curves for BC-1Cas9 ΔIRAK4 clones. (G) Quantification of colony formation in BCBL-1Cas9 ΔMYD88 stable cell lines. Colony counts were obtained using ImageJ, and the square root of the number of colonies was plotted; n = 15. (H) Quantification of colony formation in BCBL-1Cas9 ΔIRAK1 stable cell lines. (I) Quantification of colony formation in BCBL-1Cas9 ΔIRAK4 stable cell lines.

Article Snippet: We obtained three IRAK1 expression plasmids from OriGene, namely, pDD1951 (RC221544, PEL phenotype full-length IRAK1), pDD1952 (RC224107, IRAK1 isoform B), pDD1953 (RC204869, IRAK1 isoform C), and the empty vector control pDD1957 (EV, pCMV6-entry).

Techniques: Western Blot, Knock-Out, Clone Assay, Cell Counting, Plasmid Preparation, Stable Transfection

Journal: Journal of Virology

Article Title: Interleukin-1 Receptor-Associated Kinase (IRAK) Signaling in Kaposi Sarcoma-Associated Herpesvirus-Induced Primary Effusion Lymphoma

doi: 10.1128/JVI.02123-19

Figure Lengend Snippet: NF-κB activation by IL-1β is not functional in ΔMYD88 clones. (A) A Western blot for phospho-NF-κB and the IRAK pathway proteins IRAK1, IRAK4 and MYD88 in WT and ΔMYD88 BCBL-1Cas9 cells 15 min post IL-1β stimulation (1 ng/μl IL-1β). (B) Quantification of luciferase production using an NF-κB reporter assays system. Two ΔMYD88 clones and WT BCBL-1Cas9 cells were stimulated with 1 ng/μl IL-1β, or mock PBS for 24 h h following transfection, and luciferase values measured 6 h h post stimulation. Results are fold change over mock. (C) Two ΔMYD88 clones and WT BCBL-1Cas9 cells were stimulated with TNF-α (1 ng/ml), and the response was compared to mock using the same procedure as in panel B.

Article Snippet: We obtained three IRAK1 expression plasmids from OriGene, namely, pDD1951 (RC221544, PEL phenotype full-length IRAK1), pDD1952 (RC224107, IRAK1 isoform B), pDD1953 (RC204869, IRAK1 isoform C), and the empty vector control pDD1957 (EV, pCMV6-entry).

Techniques: Activation Assay, Functional Assay, Clone Assay, Western Blot, Luciferase, Transfection

Journal: Journal of Virology

Article Title: Interleukin-1 Receptor-Associated Kinase (IRAK) Signaling in Kaposi Sarcoma-Associated Herpesvirus-Induced Primary Effusion Lymphoma

doi: 10.1128/JVI.02123-19

Figure Lengend Snippet: Complementation of IRAK1 restores signaling function in KO cells. (A) Western blot in WT BCBL-1Cas9 cells showing expression of Myc-tagged IRAK1 in BCBL-1Cas9 cells. (B) IRAK expression plasmids were conucleofected with an NF-κB reporter-driven luciferase plasmid into WT or ΔIRAK1 BCBL-1Cas9 cells. Cells were stimulated with IL-1β or PBS (mock), and luciferase values were measured 6 h poststimulation. Shown are relative activities adjusted across multiple biological replicates and scales as fraction of maximal response on a log10 scale. (C) IRAK expression plasmids were conucleofected with an NF-κB reporter-driven luciferase plasmid into WT, ΔIRAK1, ΔIRAK4, or ΔMYD88 BCBL-1Cas9 cells. Cells were stimulated with IL-1β, TNF-α, or PBS (mock), and luciferase values were measured 6 h poststimulation. Shown are relative activities adjusted across multiple biological replicates and scales as fraction of maximal response on a log10 scale.

Article Snippet: We obtained three IRAK1 expression plasmids from OriGene, namely, pDD1951 (RC221544, PEL phenotype full-length IRAK1), pDD1952 (RC224107, IRAK1 isoform B), pDD1953 (RC204869, IRAK1 isoform C), and the empty vector control pDD1957 (EV, pCMV6-entry).

Techniques: Western Blot, Expressing, Luciferase, Plasmid Preparation

Journal: Journal of Virology

Article Title: Interleukin-1 Receptor-Associated Kinase (IRAK) Signaling in Kaposi Sarcoma-Associated Herpesvirus-Induced Primary Effusion Lymphoma

doi: 10.1128/JVI.02123-19

Figure Lengend Snippet: Comparison of in vitro and in culture IRAK inhibitor activity. (A) EC50 curves (growth) for three commercially available IRAK inhibitors. Fraction of response is shown on the vertical axis and concentration (in μM) on the horizontal axis. Inh1 (CAS no. 1042224-63-4), Inh4 (CAS no. 1012104-68-5), and Inh1-4 (CAS no. 509093-47-4). The EC50 value on each plot is the average from four experiments. (B) Quantification of luciferase production in cells transfected with an NF-κB-driven luciferase plasmid, incubated with inhibitor, a nd stimulated with 1 ng/μl IL-1β. Luciferase values were measured 6 h poststimulation. All values are fold change over that with mock PBS stimulation on the vertical axis and inhibitor concentration (in μM) on the horizontal axis. (C) A DiscoverX KINOMEscan analysis for each IRAK inhibitor at 250 nM. Purple or blue dots and represent IRAK4 or IRAK1 kinase, respectively. Size of the circle is proportional to percent activity inhibited by the inhibitors.

Article Snippet: We obtained three IRAK1 expression plasmids from OriGene, namely, pDD1951 (RC221544, PEL phenotype full-length IRAK1), pDD1952 (RC224107, IRAK1 isoform B), pDD1953 (RC204869, IRAK1 isoform C), and the empty vector control pDD1957 (EV, pCMV6-entry).

Techniques: In Vitro, Activity Assay, Concentration Assay, Luciferase, Transfection, Plasmid Preparation, Incubation

Journal: International journal of molecular medicine

Article Title: Xuebijing exerts protective effects on lung permeability leakage and lung injury by upregulating Toll-interacting protein expression in rats with sepsis.

doi: 10.3892/ijmm.2014.1943

Figure Lengend Snippet: Figure 1. Effect of XBJ on the mRNA expression of Tollip, IRAK1, TLR4, NF-κB65 and TRAF6 in lung tissue. Groups of mice were challenged with CLP and treated with XBJ 24 h later. The expression of Tollip, IRAK1, TLR4, NF-κB65 and TRAF6 in lung tissue was determined by RT-PCR. Representative RT-PCR shows the level of Tollip, IRAK1, TLR4, NF-κB65, and TRAF6 expression in the four rat groups. M, marker; A, normal control group; B, sham operation group; C, control group; D, treatment group.

Article Snippet: Rabbit anti-mouse Tollip, TLR4, TRAF6, p-IRAK1, VEGF-α, HO-1 and NF-κB polyclonal antibodies were purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Marker, Control

Journal: International journal of molecular medicine

Article Title: Xuebijing exerts protective effects on lung permeability leakage and lung injury by upregulating Toll-interacting protein expression in rats with sepsis.

doi: 10.3892/ijmm.2014.1943

Figure Lengend Snippet: Figure 2. Administration of XBJ led to increased expression levels of Tollip mRNA, and inhibition of TLR4, NF-κB65 and TRAF6 mRNA expression in lung tissue in CLP-ALI mice. The expression of Tollip, IRAK1, TLR4, NF-κB65 and TRAF6 in lung tissue was determined by RT-PCR. Statistical summary of the densitometric analysis of Tollip, IRAK1, TLR4, NF-κB65 and TRAF6 expression in the four rat groups. Data are presented as mean ± stan dard deviation of one experiment consisting of three replicates. Experiments were performed in triplicate; ﹡P<0.05 and ﹡﹡P<0.01 vs. normal control group and sham operation group. ﹟P<0.05 and ﹟﹟P<0.01 vs. control group.

Article Snippet: Rabbit anti-mouse Tollip, TLR4, TRAF6, p-IRAK1, VEGF-α, HO-1 and NF-κB polyclonal antibodies were purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China).

Techniques: Expressing, Inhibition, Reverse Transcription Polymerase Chain Reaction, Control

Journal: International journal of molecular medicine

Article Title: Xuebijing exerts protective effects on lung permeability leakage and lung injury by upregulating Toll-interacting protein expression in rats with sepsis.

doi: 10.3892/ijmm.2014.1943

Figure Lengend Snippet: Figure 4. Administration of XBJ enhanced the expression of Tollip protein protein, and inhibition TLR4, NF-κB65, p-IRAK1 and TRAF6 protein expression in lung tissue in CLP-ALI mice. Groups of mice were challenged with LPS and treated with salidroside 24 h later. Tollip, p-IRAK1, TLR4, NF-κB65 and TRAF6 were assayed by western blot analysis. Statistical summary of the densitometric analysis of Tollip, p-IRAK1, TLR4, NF-κB65 and TRAF6 protein expression in the four rat groups. Data are presented as mean ± standard deviation of one experiment consisting of three replicates. Experiments were performed in triplicate; ﹡﹡P<0.01 vs. the normal control group and sham operation group. ﹟P<0.05, ﹟﹟P<0.01 vs. the control group.

Article Snippet: Rabbit anti-mouse Tollip, TLR4, TRAF6, p-IRAK1, VEGF-α, HO-1 and NF-κB polyclonal antibodies were purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China).

Techniques: Expressing, Inhibition, Western Blot, Standard Deviation, Control

Journal: International journal of molecular medicine

Article Title: Xuebijing exerts protective effects on lung permeability leakage and lung injury by upregulating Toll-interacting protein expression in rats with sepsis.

doi: 10.3892/ijmm.2014.1943

Figure Lengend Snippet: Figure 3. Administration of XBJ enhanced the expression of Tollip protein, and inhibition of TLR4, NF-κB65, p-IRAK1 and TRAF6 protein expression in lung tissue in CLP-ALI mice. Groups of mice were challenged with LPS and treated with salidroside 24 h later. Tollip, p-IRAK1, TLR4, NF-κB65 and TRAF6 were assayed by western blot analysis. Representative western blots show the level of Tollip, p-IRAK1, TLR4, NF-κB65 and TRAF6 protein expression in the four rat groups. A, normal control group; B, sham operation group; C, control group; D, treatment group.

Article Snippet: Rabbit anti-mouse Tollip, TLR4, TRAF6, p-IRAK1, VEGF-α, HO-1 and NF-κB polyclonal antibodies were purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China).

Techniques: Expressing, Inhibition, Western Blot, Control