Journal: Cell reports

Article Title: The IRAK1/IRF5 axis initiates IL-12 response by dendritic cells and control of Toxoplasma gondii infection

doi: 10.1016/j.celrep.2024.113795

Figure Lengend Snippet: (A and C) Survival data from C57BL/6 mice IP infected with T. gondii Me49 (25 cysts per mouse): WT (n = 35), Irak1 −/− (n = 19), Irak2 −/− (n = 19), Irak1 −/− Irak2 −/− (n = 27), Irak4 Ki (n = 22), Irak1 −/− Irak4 Ki (n = 12), Irak2 −/− Irak4 Ki (n = 22), and Irak4 −/− (n = 8). (B) Cysts counts in the brain of WT (n = 15), Irak1 −/− (n = 14), and Irak2 −/− (n = 13) mice IP infected with T. gondii Me49 (25 cysts per mouse) for 40 days. (D–G) Quantification of IL-12 p40 and IFN-γ in the plasma (D and E) and peritoneal cavity (F and G) 5 days post IP infection with T. gondii Me49 (25 cysts per mouse) in WT (n = 13), Irak1 −/− (n = 7), Irak2 −/− (n = 14), Irak1 −/− Irak2 −/− (n = 6), Irak4 Ki (n = 12), Irak1 −/− Irak4 Ki (n = 4), Irak2 −/− Irak4 Ki (n = 14), and Irak4 −/− (n = 5) mice or mice mock-infected with PBS (WT uninfected, n = 10). (H and I) IL-12 p40 production in splenocytes unprimed (H) or primed with IFN-γ (100 ng mL −1 , 24 h) after in vitro infection with T. gondii Me49 tachyzoites at multiplicity of infection (MOI) 3 for 24 h (n = 2 for all strains). n represents the number of animals. In (A) and (C), survival data were pooled from five independent experiments. In (B) and (D)–(I), data were pooled from two (H and I), three (B), or four (D–G) independent experiments and are presented as mean ± standard error of the mean (SEM). Each independent experiment consisted of three technical replicates. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 in relation to the WT (one-way analysis of variance [ANOVA] with Tukey’s multiple-comparisons test).

Article Snippet: The following antibodies and dilutions were used: IRAK1 (Cell Signaling, 4504S, 1:1000), IRAK2 (Abcam, ab62419, 1:500), IRAK4 (Novus, NB500–597, 1:1000), MyD88 (R&D Systems, AF3109, 1:500), rodent-specific IRF5 (Cell Signaling, 4950, 1:1000), USF2 (Novus Biologicals, NBP1–92649, 1:2000), c-Rel (Cell Signaling, 67489, 1:1000), phospho-IκB-α (Ser32) (Cell Signaling, 2859, 1:750), IκB-α (Cell Signaling, 4812S, 1:1000), phospho-RelA/NF-κB (Ser 536) (Cell Signaling, 3033, 1:1000), RelA/NF-κB (Novus Biologicals, NB100–2176, 1:1000), Actin (Sigma-Aldrich, A2066, 1:3000), phospho-SAPK/JNK (Thr 183/Tyr 185) (Cell Signaling, 4668T, 1:1000), phospho-p38 MAPK (Thr 180/Tyr 182) (Cell Signaling, 4511T, 1:1000), phospho-p44/42 MAPK (Erk 1/2) (Thr 202/Tyr 204) (Cell Signaling, 4370T, 1:2000), phosphor-IKK (Ser 176/180) (Cell Signaling, 2697, 1:1000), CNBP (Santa Cruz, sc-515387-X, 1:200), anti-Goat-IgG HRP-conjugated (R&D Systems, HAF017, 1:5000), anti-Rabbit-IgG HRP-conjugated (Sigma-Aldrich, A0545, 1:5000), anti-Mouse-IgG HRP-conjugated (Cell Signaling, 7076S, 1:5000).

Techniques: Infection, In Vitro

Journal: Cell reports

Article Title: The IRAK1/IRF5 axis initiates IL-12 response by dendritic cells and control of Toxoplasma gondii infection

doi: 10.1016/j.celrep.2024.113795

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The following antibodies and dilutions were used: IRAK1 (Cell Signaling, 4504S, 1:1000), IRAK2 (Abcam, ab62419, 1:500), IRAK4 (Novus, NB500–597, 1:1000), MyD88 (R&D Systems, AF3109, 1:500), rodent-specific IRF5 (Cell Signaling, 4950, 1:1000), USF2 (Novus Biologicals, NBP1–92649, 1:2000), c-Rel (Cell Signaling, 67489, 1:1000), phospho-IκB-α (Ser32) (Cell Signaling, 2859, 1:750), IκB-α (Cell Signaling, 4812S, 1:1000), phospho-RelA/NF-κB (Ser 536) (Cell Signaling, 3033, 1:1000), RelA/NF-κB (Novus Biologicals, NB100–2176, 1:1000), Actin (Sigma-Aldrich, A2066, 1:3000), phospho-SAPK/JNK (Thr 183/Tyr 185) (Cell Signaling, 4668T, 1:1000), phospho-p38 MAPK (Thr 180/Tyr 182) (Cell Signaling, 4511T, 1:1000), phospho-p44/42 MAPK (Erk 1/2) (Thr 202/Tyr 204) (Cell Signaling, 4370T, 1:2000), phosphor-IKK (Ser 176/180) (Cell Signaling, 2697, 1:1000), CNBP (Santa Cruz, sc-515387-X, 1:200), anti-Goat-IgG HRP-conjugated (R&D Systems, HAF017, 1:5000), anti-Rabbit-IgG HRP-conjugated (Sigma-Aldrich, A0545, 1:5000), anti-Mouse-IgG HRP-conjugated (Cell Signaling, 7076S, 1:5000).

Techniques: Recombinant, Modification, Saline, Protease Inhibitor, Marker, Western Blot, Extraction, Enzyme-linked Immunosorbent Assay, Software

Journal: Journal of Leukocyte Biology

Article Title: Induction of endotoxin tolerance in vivo inhibits activation of IRAK4 and increases negative regulators IRAK-M, SHIP-1, and A20

doi: 10.1189/jlb.0611273

Figure Lengend Snippet: (A) Twenty-six hours after i.p. inoculation of PBS or LPS (25 μg/mouse), spleen were isolated and homogenized, and spleen cells were adhered to plastic to obtain macrophages, followed by stimulation of cells for 15 min with medium or 100 ng/ml LPS. IRAK4 proteins were immunoprecipitated (IP) from cell lysates and subjected to in vitro kinase assays with MBP as a substrate. Cell lysates were analyzed by immunoblotting, using anti-IκBα, anti-IRAK4, and antitubulin antibodies. The results of representative (n=3) experiments are presented. (B) Densitometric quantification of the data shown in A. Intensities of p-IRAK4 and p-MBP bands were measured and normalized to the levels of total IRAK4 and tubulin. Data in the treatment groups were divided by values obtained in medium-treated macrophages obtained from PBS-injected mice and presented as fold induction.

Article Snippet: Reagents Antibodies against IκBα, β-actin, tubulin, and A20 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-p-p38, anti-total p38, and anti-IRAK4 antibodies were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Isolation, Immunoprecipitation, In Vitro, Western Blot, Injection

Journal: Molecules

Article Title: A Novel IRAK4 Inhibitor DW18134 Ameliorates Peritonitis and Inflammatory Bowel Disease

doi: 10.3390/molecules29081803

Figure Lengend Snippet: Chemistry structure of compound DW18134 and its effects on the LPS-induced IRAK4 signaling transduction and secretion of cytokines in macrophages. ( A ) Chemical structure of DW18134. ( B ) Representative immunoblots of p-IRAK4 and p-IKK in PCM cells that were stimulated with LPS (5 µg/mL) and treated with or without indicated concentrations of DW18134 for 2 h. ( C ) Quantification of p-IRAK4 and p-IKK levels in PCM cells. ( D ) Representative immunoblots of p-IRAK4 and p-IKK in RAW264.7 cells that were stimulated with LPS and treated with or without DW18134. ( E ) p-IRAK4 and p-IKK levels in RAW264.7 cells were quantified. ( F , G ) Secretion levels of TNF-a and IL-6 in the supernatants of PCM and RAW264.7 cells were measured by ELISA. ( H , I ) Following the treatment of DW18134 for 24 h, cell viability of PCM and RAW 264.7 cells was determined by CCK-8 assays. A representative of three independent repeats is shown in ( B , D ). Data represent the mean ± standard deviation (SD) from three independent biological replicates in ( C , E – I ). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. LPS; ### p < 0.001 vs. control. ns: no significance.

Article Snippet: The antibody for Phospho-IRAK4 (Thr345/Ser346) (D6D7) Rabbit mAb(dilution, 1:1000; #11927), IRAK4 antibody (dilution, 1:1000; #4363), Phospho-IKKα/β (Ser176/180) (16A6) Rabbit mAb(dilution, 1:1000; #2697), NF-κB p65 (D14E12) Rabbit mAb(dilution, 1:1000; #8242), Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb(dilution, 1:1000; #3033), and actin (dilution, 1:20,000; # 60008-1-Ig, Proteintech, Cranbury, NJ, USA) were purchased from Cell Signaling Technology (CST, Boston, MA, USA).

Techniques: Transduction, Western Blot, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Standard Deviation, Control

Journal: Molecules

Article Title: A Novel IRAK4 Inhibitor DW18134 Ameliorates Peritonitis and Inflammatory Bowel Disease

doi: 10.3390/molecules29081803

Figure Lengend Snippet: The IC 50 values of compounds against IRAK4.

Article Snippet: The antibody for Phospho-IRAK4 (Thr345/Ser346) (D6D7) Rabbit mAb(dilution, 1:1000; #11927), IRAK4 antibody (dilution, 1:1000; #4363), Phospho-IKKα/β (Ser176/180) (16A6) Rabbit mAb(dilution, 1:1000; #2697), NF-κB p65 (D14E12) Rabbit mAb(dilution, 1:1000; #8242), Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb(dilution, 1:1000; #3033), and actin (dilution, 1:20,000; # 60008-1-Ig, Proteintech, Cranbury, NJ, USA) were purchased from Cell Signaling Technology (CST, Boston, MA, USA).

Techniques:

Journal: Molecules

Article Title: A Novel IRAK4 Inhibitor DW18134 Ameliorates Peritonitis and Inflammatory Bowel Disease

doi: 10.3390/molecules29081803

Figure Lengend Snippet: DW18134 reduced macrophage infiltration and pro-inflammatory gene expression and abrogated IRAK4 signaling pathway activation in LPS-induced peritonitis mice ( n = 8). Immunohistochemical staining of macrophage infiltration in the liver of mice with peritonitis. Includes quantitative data ( A ) and representative images ( B ). ( C ) Tnfa , Ill6 , and Il1b gene expression from liver homogenates measured by RT-qPCR. ( D ) Expression of p-IRAK4, p-IKK, and p-p65 in liver tissue of peritonitis mice treated with DW18134 or PF-06650833. ( E ) Quantification of p-IRAK4, p-IKK, and p-p65 levels in liver tissue based on three independent biological replicates. Data are expressed as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. LPS; ### p < 0.001 vs. control. ns: no significance.

Article Snippet: The antibody for Phospho-IRAK4 (Thr345/Ser346) (D6D7) Rabbit mAb(dilution, 1:1000; #11927), IRAK4 antibody (dilution, 1:1000; #4363), Phospho-IKKα/β (Ser176/180) (16A6) Rabbit mAb(dilution, 1:1000; #2697), NF-κB p65 (D14E12) Rabbit mAb(dilution, 1:1000; #8242), Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb(dilution, 1:1000; #3033), and actin (dilution, 1:20,000; # 60008-1-Ig, Proteintech, Cranbury, NJ, USA) were purchased from Cell Signaling Technology (CST, Boston, MA, USA).

Techniques: Gene Expression, Activation Assay, Immunohistochemical staining, Staining, Quantitative RT-PCR, Expressing, Control

Journal: Molecules

Article Title: A Novel IRAK4 Inhibitor DW18134 Ameliorates Peritonitis and Inflammatory Bowel Disease

doi: 10.3390/molecules29081803

Figure Lengend Snippet: DW18134 reduced macrophage infiltration and the expression of inflammatory factors and restored the intestinal barrier in mice with DSS ( n = 6). ( A , B ) Immunohistochemistry was used to detect macrophage infiltration in the colorectum. ( C , D ) Gene expression levels of tight junction proteins and Mucin2 were detected in the colon of IBD mice by RT-qPCR. ( E ) The mRNA expression of cytokines Tnfa , Il6 , and Il1b in colonic tissues. ( F ) Representative immunoblots of p-IRAK4 and p-IKK in the colonic homogenates from each group. p-IRAK4 and p-IKK levels were quantified (right panel). Quantified data represent the mean ± SD from three independent biological replicates. * p < 0.05, ** p < 0.01 vs. 3.5%DSS; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. control. ns: no significance.

Article Snippet: The antibody for Phospho-IRAK4 (Thr345/Ser346) (D6D7) Rabbit mAb(dilution, 1:1000; #11927), IRAK4 antibody (dilution, 1:1000; #4363), Phospho-IKKα/β (Ser176/180) (16A6) Rabbit mAb(dilution, 1:1000; #2697), NF-κB p65 (D14E12) Rabbit mAb(dilution, 1:1000; #8242), Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb(dilution, 1:1000; #3033), and actin (dilution, 1:20,000; # 60008-1-Ig, Proteintech, Cranbury, NJ, USA) were purchased from Cell Signaling Technology (CST, Boston, MA, USA).

Techniques: Expressing, Immunohistochemistry, Gene Expression, Quantitative RT-PCR, Western Blot, Control

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

Article Title: Interacting neuroendocrine and innate and acquired immune pathways regulate neutrophil mobilization from bone marrow following hemorrhagic shock.

doi: 10.4049/jimmunol.182.1.572

Figure Lengend Snippet: FIGURE 3. Pretreatment with neutralizing Ab to HMGB1 prevents HS/ R-induced CD11bGr-1 cells mobilization from bone marrow and TLR4 signaling activation. WT mice received anti-HMGB1 Ab (600 g per mouse) or nonimmune control IgG by i.p. injection 10 min before HS/R or sham operation. A, Femoral bones from the mice were harvested at 4 h after HS/R or sham operation, and bone marrow cells were then collected for detection of CD11bGr-1 cells by flow cytometry. Representative density plots of bone marrow CD11bGr-1 cells are shown. Results show mean and SE of the changes in bone marrow CD11bGr-1 cells from n three mice. , p 0.01 compared with other groups. B, Blood PMN were isolated from the mice at 2 h after HS/R or sham operation for detection of IRAK4 activity. Results show mean and SE of the changes in IRAK4 activity from n 3 mice. , p 0.01 compared with other groups.

Article Snippet: Polyclonal rabbit anti-IRAK4 Ab and MyD88 homodimerization inhibitory peptide set were purchased from Imgenex.

Techniques: Activation Assay, Control, Injection, Cytometry, Isolation, Activity Assay

Journal: Cell Reports Medicine

Article Title: Osteoclast-derived apoptotic bodies inhibit naive CD8 + T cell activation via Siglec15, promoting breast cancer secondary metastasis

doi: 10.1016/j.xcrm.2023.101165

Figure Lengend Snippet: Siglec15 inhibits naive CD8 + T cell activation via blocking TLR2 co-stimulatory signaling pathways (A) Representative flow cytometry results of CD8 + T cell (stained with CFSE) exposed to different treatments. (B) Quantification of CD8 + T cell division and immune secretion (IFN-γ, TNF-α, IL-2; per well of 96-well plate), n = 3. (C) Western blot result showed expression of IRAK4, TRAF6, and TAK1 in CD8 + T cell lysate after different treatments, and quantification of the relative expression of TRAF6 in CD8 + T cells under different treatments, n = 3. (D) Representative IHC for IRAK4, TRAF6, and TAK1 in bone metastases sections from three groups of mice. Scale bar: 200 μm. (E) Semi-quantitative analysis of IRAK4, TRAF6, and TAK1 IHC staining images, n = 5. The data in each panel represent the means ± SD and were analyzed by Student’s two-tailed unpaired t test, ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: Rabbit anti-IRAK4 , Bioss , Cat#bs-2440R;RRID: AB_11067218.

Techniques: Activation Assay, Blocking Assay, Flow Cytometry, Staining, Western Blot, Expressing, Immunohistochemistry, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Osteoclast-derived apoptotic bodies inhibit naive CD8 + T cell activation via Siglec15, promoting breast cancer secondary metastasis

doi: 10.1016/j.xcrm.2023.101165

Figure Lengend Snippet:

Article Snippet: Rabbit anti-IRAK4 , Bioss , Cat#bs-2440R;RRID: AB_11067218.

Techniques: Virus, Recombinant, Plasmid Preparation, Staining, Cell Isolation, CCK-8 Assay, Enzyme-linked Immunosorbent Assay, Purification, Bicinchoninic Acid Protein Assay, Isolation, Software