Journal: Experimental Dermatology

Article Title: NLRP3 inflammasome activation contributes to development of alopecia areata in C3H/HeJ mice

doi: 10.1111/exd.14432

Figure Lengend Snippet: IFN‐α signal pathway is activated in lesions in the initial phase of alopecia areata. IFN‐α levels in serum (A) and skin (B) from naive, preonset, initial AA and severe AA mice were measured by ELISA. mRNA expressions of IRF7 (C), IRAK4 (D), TRAF3 (E), HMGB1 (F), RAGE (G) and HSP70 (H) were assessed by RT‐PCR. (I) Serum HSP70 level was measured by ELISA. (J) Expression of mmu ‐ miR ‐ 30b ‐ 3p in blood was assessed using RT‐PCR. Skin samples were collected from non‐lesional areas (NL; normal hairy area) and lesions (L; hair loss area). Data are expressed as mean ± SE ( N = 4). ‡ , p < 0.05. ‡‡ , p < 0.01. vs. naive (Dunnett's multiple comparison test). Abbreviations: AA, alopecia areata; ELISA, enzyme‐linked immunosorbent assay; RT‐PCR, reverse transcription polymerase chain reaction; SE, standard error; and N.D., not detected

Article Snippet: The primer sets purchased from Thermo Fisher Scientific were as follows: Ager (Mm01134790_g1), Casp1 (Mm00438023_m1), Cxcl9 (Mm00434946_m1), Cxcl10 (Mm00445235_m1), Cxcl11 (Mm00444662_m1), Gzmb (Mm00442837_m1), Hspa1a (Mm01159846_s1), Hmgb1 (Mm00849805_gH), Ifng (Mm01168134_m1), Irak4 (Mm00459443_m1), Irf7 (Mm00516793_g1), Nlrp3 (Mm00840904_m1), Pycard (Mm00445747_g1), Traf3 (Mm00495752_m1) and Gapdh (Mm99999915_g1).

Techniques: Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Comparison, Reverse Transcription, Polymerase Chain Reaction

Journal: Journal of Lipid Research

Article Title: PKCδ-IRAK1 axis regulates oxidized LDL-induced IL-1β production in monocytes [S]

doi: 10.1194/jlr.M045658

Figure Lengend Snippet: IRAK1 and IRAK4 mediate IL-1β production in THP1 cells. Ox-LDL-induced (40 μg/ml) IL-1β production at 48 h in THP1 cells was measured in triplicate after pretreatment with IRAK1/4 INH (0.3 μM, n = 6) (A), IRAK1 siRNA (3 μg, n = 4) (B), IRAK2 siRNA (3 μg, n = 4) (C), IRAK3 siRNA (3 μg, n = 4) (D), and IRAK4 siRNA (3 μg, n = 4) (E). Control siRNA (3 μg) transfected cells were used as negative control. Culture supernatant was used to measure IL-1β by ELISA, while a portion of cells was lysed and analyzed by Western blotting for the expression of IRAK1, -2, -3, -4, and β-actin. Blots represent one of four similar experiments. Values represent mean ± SE. ***P < 0.001 versus control; #P < 0.05, ###P < 0.001 versus Ox-LDL alone.

Article Snippet: IRAK1, IRAK4, phospho-IRAK, phospho-PKCδ, and PKCδ antibodies were also procured from Cell Signaling Technology (Danvers, MA).

Techniques: Control, Transfection, Negative Control, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing

Journal: Molecules (Basel, Switzerland)

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

doi: 10.3390/molecules29081803

Figure Lengend Snippet: Figure 1. 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 stimu- lated 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) Fol- lowing 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 (Basel, Switzerland)

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

doi: 10.3390/molecules29081803

Figure Lengend Snippet: Figure 3. DW18134 reduced macrophage infiltration and pro-inflammatory gene expression and abrogated IRAK4 signaling pathway activation in LPS-induced peritonitis mice (n = 8). Immunohisto- chemical 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, Staining, Quantitative RT-PCR, Expressing, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: TRAF6-mediated ubiquitination of MST1/STK4 attenuates the TLR4-NF-κB signaling pathway in macrophages

doi: 10.1007/s00018-020-03650-4

Figure Lengend Snippet: MST1/STK4 negatively regulates LPS-induced NF-κB signaling in BMDMs. a BMDMs isolated from WT and MST1/STK4−/− mice were exposed to LPS (1 μg/ml) for the indicated times in culture and then assayed for IL-6 and TNF-α mRNA by quantitative RT-PCR analysis. Data are representative of three independent experiments (means ± SD of triplicate samples). *p < 0.05. b BMDMs were incubated in the absence or presence of LPS (1 μg/ml) for 12 h, after which IL-6 and TNF-α released into culture media were quantified by ELISA. Data are means ± SD from three independent experiments. *p < 0.05. c BMDMs exposed to LPS (1 μg/ml) for the indicated times were subjected to immunoblot analysis with antibodies to phospho-p65 (Ser536), to p65, to IκBα, to MST1/STK4, or to GAPDH. The intensity of bands corresponding to phospho-p65 was quantified by densitometry, and normalized by that of those for p65. The relative values of the intensity from five independent experiments were shown below. *p < 0.05. NS not significant. d BMDMs exposed to LPS (1 μg/ml) for the indicated times were immunoprecipitated with antibodies to TRAF6 or to MST1/STK4. The TRAF6 precipitates were examined by immunoblot analysis (IB) with anti-ubiquitin antibody, whereas the MST1/STK4 precipitates were subjected to an immune complex kinase assay with myelin basic protein (MyBP) as substrate. The relative values for MST1/STK4 activity are shown. Cell lysates were also immunoblotted directly with antibodies to phospho-IKKα/β (Ser176/180), to IKKα/β, to phospho-TAK1 (Thr184/187), to TAK1, to TRAF6, to MST1/STK4, or to GAPDH. The intensity of the bands for ubiquitinated TRAF6 [TRAF6-Ub(n)], phospho-IKKα/β, or phospho-TAK1 normalized by that of those for the corresponding total proteins was quantified by densitometry, and the relative values of the intensity were shown. Quantitative data are mean ± SD from three independent experiments. *p < 0.05. NS not significant. e BMDMs exposed to LPS (1 μg/ml) for the indicated times were immunoblotted with antibodies to phospho-IRAK1 (Thr209), to IRAK1, to phospho-IRAK4 (Thr345/Ser346), to IRAK4, to MST1/STK4, or to GAPDH. The intensity of the bands for phospho-IRAK1 or phospho-IRAK4 normalized by that of those for the corresponding total proteins was quantified by densitometry, and the relative values of the intensity were shown. Quantitative data are mean ± SD from three independent experiments. NS not significant

Article Snippet: Antibodies and reagents Rabbit polyclonal antibodies to MST1/STK4 and to TAK1 as well as rabbit monoclonal antibodies to phospho-MST1/STK4(Thr 183 ), phospho-IKKα/β (Ser 176/180 ), to phospho-TAK1 (Thr 184/187 ), to phospho-p65 (Ser 536 ), to phospho-IκBα Ser 32/36 , to phospho-IRAK4 (Thr 345 /Ser 346 ), to IRAK4, to IRAK1, to IKKβ, and to TRAF6 were obtained from Cell Signaling Technology (Boston, MA).

Techniques: Isolation, Quantitative RT-PCR, Incubation, Enzyme-linked Immunosorbent Assay, Western Blot, Immunoprecipitation, Ubiquitin Proteomics, Immune Complex Kinase Assay, Activity Assay

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

Article Title: IFN-beta1a inhibits the secretion of Th17-polarizing cytokines in human dendritic cells via TLR7 up-regulation.

doi: 10.4049/jimmunol.0802226

Figure Lengend Snippet: FIGURE 2. IFN-1a treatment up-regulates TLR7 and its signaling pathway, while it down-regulates the expression of IL-1R and IL-1/. A, Three 106 DCs per condition derived from 10 RR MS patients were pretreated with IFN-1a for 24 h and then matured with LPS for 2 h before RNA extraction. TLR7, MyD88, IRAK4, TRAF6, IL-1R1, IL-1, and IL-1 gene expression was measured by RT- PCR. The results are presented as relative gene expression normalized for the 18S RNA expression. B, Three 106 DCs per condition from 16 RR MS patients were pretreated with IFN-1a for 24 h and then matured with LPS for 48 h. DCs were stained with PE-Cy-CD11c, allophycocyanin-CD1a mAb, and anti-IL-1R1 Ab, followed by FITC-conjugated secondary Ab, anti-TLR7 primary Ab, and FITC-conjugated secondary Ab. FITC-conjugated IgG isotype Ab was used as a control. The percentage of positive cells was determined in the CD1a-gated population. C, Three 106 DCs per condition derived from three RR MS patients were pretreated by IFN-1a for 24 h and then matured with LPS for 5 h before protein extraction. The expression of TLR7, MyD88, IRAK4, TRAF6, IL-1R1, and -actin were measured by Western blotting. The results present one of three similar experiments.

Article Snippet: Nonspecific binding was blocked for 1 h in the blocking buffer (10 mM Tris (pH 7.5), 150 mM NaCl, 2% fish gelatin, and 1% OVA in double-distilled water) and incubated overnight with rabbit anti-human Ab for TLR7 (Abcam), MyD88, TRAF6 (Santa Cruz Biotechnology), and IRAK4 (ProSci) and with mouse anti-human Ab for IL1R1 and -actin (Santa Cruz Biotechnology).

Techniques: Expressing, Derivative Assay, RNA Extraction, Gene Expression, Reverse Transcription Polymerase Chain Reaction, RNA Expression, Staining, Control, Protein Extraction, Western Blot

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

Article Title: IFN-beta1a inhibits the secretion of Th17-polarizing cytokines in human dendritic cells via TLR7 up-regulation.

doi: 10.4049/jimmunol.0802226

Figure Lengend Snippet: FIGURE 4. IFN-1a modifies MyD88/IRAK4/ TRAF6 signaling and cytokine production in DCs through its induction of TLR7 expression. Three 106/well DCs generated from six RR MS patients were plated in antibiotic-free medium. After a 24-h incubation at 37oC, the DCs were transfected with TLR7 siRNA or control siRNA. The siRNA-treated cells were harvested and cultured in serum-free me- dium in the absence or presence of IFN-1 for 24 h, followed by LPS maturation for 5 h before the pro- tein extraction, and for 48 h before the SN collec- tion. A, The expression of TLR7, MyD88, IRAK4, TRAF6, IL-1R1, and -actin was measured by Western blotting. The results present one of three similar experiments. B, The production of IL-1, TGF-1, IL-23/p19, and IL-27 was measured by ELISA. The results present six experiments per- formed in triplicate. Statistical analysis was per- formed using a repeated measures ANOVA. , p 0.05; , p 0.01; and , p 0.001.

Article Snippet: Nonspecific binding was blocked for 1 h in the blocking buffer (10 mM Tris (pH 7.5), 150 mM NaCl, 2% fish gelatin, and 1% OVA in double-distilled water) and incubated overnight with rabbit anti-human Ab for TLR7 (Abcam), MyD88, TRAF6 (Santa Cruz Biotechnology), and IRAK4 (ProSci) and with mouse anti-human Ab for IL1R1 and -actin (Santa Cruz Biotechnology).

Techniques: Expressing, Generated, Incubation, Transfection, Control, Cell Culture, Extraction, Western Blot, Enzyme-linked Immunosorbent Assay

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: Rabbit pAb anti-IRAK4 , Novus , NB500–597; RRID AB_2943026.

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