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: 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