Journal: Frontiers in Immunology

Article Title: Lack of Association Between Sex Hormones, MDSCs, LDGs and pDCs in Males and Females With Systemic Lupus Erythematosus

doi: 10.3389/fimmu.2022.888501

Figure Lengend Snippet: IRF7 expression is upregulated in SLE, independent of pDC expression. RNA was isolated from paxgene blood samples and RT-PCR was run using Taqman probes for signaling molecules within the TLR activation pathway: (A) IRF5 , (B) IRF7 , and (C) IRAK4 . A single HC male sample was set as the standard level of expression and all samples were quantified as the relative gene expression to this control. Each sample was then normalized to its %pDCs as determined by flow cytometry analysis as presented in Figure 1B . Comparisons were made using Kruskal-Wallis Test with Dunn’s test for multiple comparisons. Bars represent median and interquartile range. aSLE F (N = 15), iSLE F (N = 50), HC F (N = 14), aSLE M (N = 5), iSLE M (N = 6), HC M (N = 5). *p < 0.05; **p < 0.01.

Article Snippet: The following TaqmanTM primer sets (ThermoFisher Scientific, Waltham, MA) were used: IFI27 (Hs01086370_m1), IFI44L (Hs00199115_m1), IFIT1 (Hs00356631_g1), ISG15 (Hs00192713_m1), RSAD2 (Hs01057264_m1), SIGLEC1 (Hs00988063_m1), IRAK4 (Hs00211610_m1), IRF5 (Hs00158114_m1), IRF7 (Hs01014809_g1), 18S (Hs999999001_s1).

Techniques: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Activation Assay, Gene Expression, Control, Flow Cytometry

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: 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