Journal: Oncotarget

Article Title: IRAK1 is a novel DEK transcriptional target and is essential for head and neck cancer cell survival

doi:

Figure Lengend Snippet: A. UMSCC1 and UMSCC47 were lentivirally depleted for DEK, as confirmed by western blot analysis, and mRNA from these lines was submitted for RNA-Sequencing (RNA-Seq). B. GeneSpring NGS analysis was performed on genes differentially expressed 1.4 fold or greater (DEKsh/NTsh). GeneSpring derived Venn diagrams depict genes altered in both cell lines and overlap highlights genes common to both UMSCC1 and UMSCC47. C. Overlapping genes (from panel B, 2282) from UMSCC1 and UMSCC47 were analyzed using ToppGene server to identify significantly altered biological processes. The top 10 biological processes are represented. D. A portion of the down-regulated gene networks analyzed on NetWalker highlights nodes containing IRAK1 and DEK in UMSCC1 cells. A full view of view of this map can be found in .

Article Snippet: TaqMan probes were as follows: DEK (Hs01078267_m1), IRAK1 (Hs01018347_m1), and GAPDH (Hs02758991_g1).

Techniques: Western Blot, RNA Sequencing, Derivative Assay

Journal: Oncotarget

Article Title: IRAK1 is a novel DEK transcriptional target and is essential for head and neck cancer cell survival

doi:

Figure Lengend Snippet: A. Table depicts fold changes for DEK and IRAK1 in UMSCC1 and UMSCC47 from GeneSpring NGS analysis. B, C, D. IRAK1 mRNA is reduced following DEK depletion. IRAK1 and DEK mRNA levels were confirmed by TaqMan qRT-PCR to validate RNA-Seq results in three cell lines: UMSCC1, UMSCC47, and UMSCC6 (HPV negative). Experiments were performed twice and standard deviation (SD) depicted. E. Western blot analysis confirms IRAK1 protein levels and downstream signaling are also depleted in the absence of DEK. Actin was used as a loading control.

Article Snippet: TaqMan probes were as follows: DEK (Hs01078267_m1), IRAK1 (Hs01018347_m1), and GAPDH (Hs02758991_g1).

Techniques: Quantitative RT-PCR, RNA Sequencing, Standard Deviation, Western Blot, Control

Journal: Oncotarget

Article Title: IRAK1 is a novel DEK transcriptional target and is essential for head and neck cancer cell survival

doi:

Figure Lengend Snippet: A. cBioPortal analysis of the TCGA HNSCC database reveals IRAK1 alterations occur in 14% of HNSCC. A total of 279 samples were analyzed and were further broken down into HPV − and HPV + subsets. B. IRAK1 is expressed in primary HNSCC tissues. CCHMC-HNSCC1 (HPV+) and CCHMC-HNSC18 (HPV-) were stained for IRAK1 by immunohistochemistry, with intense staining in both the nucleus and cytoplasm. n = 4 samples were stained. Images were taken at 5x and 20x magnification. C. IRAK1 staining is not expressed in well differentiated layers of normal human skin (NHSK) from unrelated donors. Images were taken at 20x magnification.

Article Snippet: TaqMan probes were as follows: DEK (Hs01078267_m1), IRAK1 (Hs01018347_m1), and GAPDH (Hs02758991_g1).

Techniques: Staining, Immunohistochemistry

Journal: Oncotarget

Article Title: IRAK1 is a novel DEK transcriptional target and is essential for head and neck cancer cell survival

doi:

Figure Lengend Snippet: A. IRAK1 loss attenuates activation of downstream signaling pathways in HNSCC. UMSCC1 and UMSCC47 were transduced with control (NTsh) or IRAK1 knockdown (IRAK1sh) vectors and protein was collected following selection in puromycin. Whole cell lysates were analyzed by western blot analysis to confirm IRAK1 knockdown, along with reduction in IRAK1 activation (pIRAK1Thr209) and MAPK pathways. GAPDH was used as a loading control. B. IRAK1-inhibitor reduces IRAK1 activation in HPV − and HPV + cell lines. UMSCC1 and UMSCC47 cells were plated and DMSO (control) or IRAK1-inhibitor was added the following day at 1 μM or 10 μM final concentrations. Cells were then collected for western blot analysis at indicated time-points. Inhibition of IRAK1 was confirmed by western blot analysis, as measured by phosphorylation of IRAK1. GAPDH was used as a loading control. C and D. TRAF6 ubiquitination is reduced following IRAK1 loss. Immunoprecipitation was performed on RIPA lysates with the TRAF6 antibody. Western blot was then performed for ubiquitin. Remaining whole cell lysates were analyzed by western blot for TRAF6, IRAK1, and GAPDH. E and F. IRAK1 loss increases cellular death via apoptosis. Cells were analyzed by flow cytometry for cleaved-caspase 3 conjugated to FITC. Experiments were performed in triplicate with SEM depicted. G. IRAK1-inhibitor increases apoptosis. UMSCC1 cells were plated and DMSO or IRAK1-inhibitor was added after cells attached. Cells and media were collected 72 hours later and analyzed for cleaved caspase-3 by flow cytometry. Experiment was performed three times, with SEM depicted. (*= p < .05).

Article Snippet: TaqMan probes were as follows: DEK (Hs01078267_m1), IRAK1 (Hs01018347_m1), and GAPDH (Hs02758991_g1).

Techniques: Activation Assay, Protein-Protein interactions, Transduction, Control, Knockdown, Selection, Western Blot, Inhibition, Phospho-proteomics, Ubiquitin Proteomics, Immunoprecipitation, Flow Cytometry

Journal: Oncotarget

Article Title: IRAK1 is a novel DEK transcriptional target and is essential for head and neck cancer cell survival

doi:

Figure Lengend Snippet: A. IRAK1 overexpression rescues ERK1/2 signaling. Sorted control or IRAK1 overexpressing cells were transduced with control (NTsh) or DEK knockdown (DEKsh) vector. After selection was complete protein was collected and analyzed by western blot. GAPDH was used as a loading control. Growth curves of control (iG2) versus IRAK1 overexpressing cells can be found in . B. IRAK1 and DEK regulate cell growth and viability independently. Cells from (A) were used to analyze apoptosis (B) cellular cycle (C) and cellular proliferation. (D). IRAK1 overexpression did not rescue the phenotypes observed with DEK loss. Experiments were performed twice and SD is represented. E. Combined IRAK1 and DEK loss increases cell death. Control and IRAK1 knockdown cells were transduced with control (AdGFP) or DEK knockdown (AdDEKsh) adenovirus. Three days post-adenoviral infection cells were collected and later analyzed for cleaved caspase-3 by flow cytometry. Graph represents fold change compared to NTsh AdGFP samples. Experiments were performed twice and SD represented. F. Molecular model. IRAK1 is a novel target in HNSCC. DEK is required for efficient expression of IRAK1 and DEK and IRAK1 also independently contribute to HNSCC cell survival and depletion of either promotes apoptosis and this may be a result of ERK1/2, p38 and/or NF-κB signaling.

Article Snippet: TaqMan probes were as follows: DEK (Hs01078267_m1), IRAK1 (Hs01018347_m1), and GAPDH (Hs02758991_g1).

Techniques: Over Expression, Control, Transduction, Knockdown, Plasmid Preparation, Selection, Western Blot, Infection, Flow Cytometry, Expressing

Journal: Cell reports

Article Title: Nanoparticle-based itaconate treatment recapitulates low-cholesterol/low-fat diet-induced atherosclerotic plaque resolution

doi: 10.1016/j.celrep.2024.114911

Figure Lengend Snippet:

Article Snippet: anti-mouse phospho-IRAK1 (Thr387) Polyclonal Antibody , Bioss Antibodies , Cat#: bs-3194R; RRID:AB_10857144.

Techniques: Purification, Plasmid Preparation, Produced, Recombinant, Concentration Assay, Saline, Labeling, Membrane, Enzyme-linked Immunosorbent Assay, Quantitation Assay, Phospholipid Assay, BIA-KA, RNAscope, HD Assay, Polymer, Sequencing, Expressing, Software, Microscopy

Journal: EMBO Molecular Medicine

Article Title: Down-regulation of BRCA1 expression by miR-146a and miR-146b-5p in triple negative sporadic breast cancers

doi: 10.1002/emmm.201100136

Figure Lengend Snippet: Relative luciferase activity after cotransfection into HeLa cells of the Luc- BRCA1 3′UTR reporter vector and of an empty miR-Vec construct (control vector), or of miR-Vec constructs expressing different miRNAs, as indicated. Error bars represent standard error of the mean (SEM) of four independent experiments. * p < 0.05; *** p < 0.001 (Student's t -test). Sequence alignment of miR-146a and miR-146b-5p and their complementary site in the schematically represented BRCA1 3′UTR. The seed sequence is bolded. Repression of luciferase activity after cotransfection into HeLa cells of the wild-type (wt) or mutated (mut146) Luc- BRCA1 3′UTR reporter vector and of control or miR-146 synthetic precursors, as indicated. Error bars represent SEM of four independent experiments. Western blot analysis with an antibody against IRAK1 or BRCA1 of proteins extracted from HeLa cells transfected with a control, miR-146a, miR-146b-5p or both miR-146a and miR-146b-5p precursors. The bands corresponding to BRCA1 were quantified relative to the α-tubulin loading control (BRCA1 normalized level) using the GelDoc™XR+ Imager (Bio-Rad) and the Image Lab™ software. The results shown are representative of at least three independent experiments.

Article Snippet: Membranes were blocked in 5% milk-TBS Tween 20% and incubated overnight with the #4359 rabbit polyclonal anti-IRAK1 antibody (Cell Signaling Technology Inc., Danvers, MA), the OP107 mouse monoclonal anti-BRCA1 antibody (Calbiochem, Darmstadt, Germany) or the T5168 mouse monoclonal anti-α-tubulin antibody (Sigma–Aldrich).

Techniques: Luciferase, Activity Assay, Cotransfection, Plasmid Preparation, Construct, Control, Expressing, Sequencing, Western Blot, Transfection, Software

Journal: EMBO Molecular Medicine

Article Title: Down-regulation of BRCA1 expression by miR-146a and miR-146b-5p in triple negative sporadic breast cancers

doi: 10.1002/emmm.201100136

Figure Lengend Snippet: Relation between BRCA1 or IRAK1 status and expression levels of miR-146a/b-5p determined by quantitative RT-PCR

Article Snippet: Membranes were blocked in 5% milk-TBS Tween 20% and incubated overnight with the #4359 rabbit polyclonal anti-IRAK1 antibody (Cell Signaling Technology Inc., Danvers, MA), the OP107 mouse monoclonal anti-BRCA1 antibody (Calbiochem, Darmstadt, Germany) or the T5168 mouse monoclonal anti-α-tubulin antibody (Sigma–Aldrich).

Techniques: Expressing

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: Nature immunology

Article Title: Essential role for the prolyl isomerase Pin1 in Toll-like receptor signaling and type I interferon-mediated immunity

doi: 10.1038/ni.2069

Figure Lengend Snippet: (a) Proteomic identification of IRAK1 as a TLR-induced Pin1 binding protein. THP1 cells stimulated with R-848 for 45 min were lysed and subjected to GST-Pin1 pulldown followed by SDS-PAGE and colloidal CBB staining. Specific GST-Pin1 interacting bands were excised and 7 peptides were identified to IRAK1 by LC-MS . (b) TLR-dependent interaction between Pin1 and IRAK1, assayed by GST-Pin1 pulldown. RAW264.7 cells stimulated with PBS or either R-848 or CpG for 30 min were subjected to immunoblotting analysis using IRAK1 antibodies after pulldown with GST or GST-Pin1. (c) TLR-dependent interaction between endogenous Pin1 and IRAK1, assayed by Co-IP. THP1 cells were stimulated with poly(I:C), R-848 or CpG and subjected to immunoprecipitation with anti-Pin1 antibodies or control IgG, followed by immunoblotting with IRAK1 antibodies. (d) The IRAK1-Pin1 interaction is sensitive to phosphatase treatment. TLR7-HEK293T cells were transfected with FLAG-IRAK1 and stimulated with R-848 and lysates were untreated or treated with calf intestinal phosphatase (CIP) phosphatase for 60 min at 30°C, followed by GST-Pin1 pulldown experiments. (e) The Pin1-IRAK1 interaction is dependent on the intrinsic kinase activity of IRAK1. FLAG-KD-IRAK1, either alone or in combination with IRAK1 were expressed in IRAK1-deficient 293T cells, followed by GST pulldown experiments (f) Pin1 binds directly to phosphorylated WT IRAK1, but not KD IRAK1. FLAG-IRAK1 and FLAG-KD IRAK1 were expressed in IRAK1-deficient 293T cells and purified using FLAG-agarose, followed by Far-Western analysis using GST-Pin1 WW domain to detect Pin1 binding using anti-GST antibody. Membranes were re-probed with FLAG antibody as a control. (g) Pin1 binds to activated WT IRAK1, but not KD IRAK1 in MEFs. FLAG-IRAK1 and its KD mutant were expressed in MEFs using retroviral infection and then treated with R-848 or control buffer, followed by GST pulldown experiments. (h) Multiple pSer-Pro motifs in the undetermined domain (UD) of IRAK1 are required for Pin1 binding. FLAG-IRAK1 and its mutants were expressed in MEFs using retroviral infection, and then treated with R-848 or control buffer, followed by GST pulldown experiments. ( i ) S173 phosphorylation of IRAK1 is induced upon TLR7 and TLR9 stimulation. THP1 cells were stimuylated with CpG or R-848 and thereafter intracellularly stained with an anti-pS173 antibody, followed by a secondary FITC conjugated antibody. Fluorescence was measured by flow cytometry.

Article Snippet: Phosphorylated Ser173-specific IRAK1 antibodies were raised by immunizing rabbits with a KLH-coupled phosphorylated Ser173-containing IRAK1 peptide (Proteintech Group) and were affinity purified, as described .

Techniques: Binding Assay, SDS Page, Staining, Liquid Chromatography with Mass Spectroscopy, Western Blot, Co-Immunoprecipitation Assay, Immunoprecipitation, Control, Transfection, Activity Assay, Purification, Mutagenesis, Retroviral, Infection, Phospho-proteomics, Fluorescence, Flow Cytometry

Journal: Nature immunology

Article Title: Essential role for the prolyl isomerase Pin1 in Toll-like receptor signaling and type I interferon-mediated immunity

doi: 10.1038/ni.2069

Figure Lengend Snippet: (a) Pin1 KO completely blocks IRAK1 activation in mouse cells following TLR7 stimulation. Pin1 WT and KO Flt3-derived pDCs (bottom) or TLR7-expressing MEF cells (top) were simulated with R-848 for the indicated times and analyzed for the characteristic IRAK1 shift by immunoblotting with IRAK1 antibodies, with IRAK4 and Pin1 amounts as controls. (b) Pin1 KO completely blocks activation of IRAK1, but not IRAK4 following TLR7 stimulation. Peritoneal macrophages from Pin1 WT and KO mice were stimulated with R-848 for the indicated times and kinase activity of IRAK1 and IRAK4 was assessed by an IP kinase autophosphorylation assay. IRAK1, IRAK4 and Pin1 protein were assayed as controls. (c) Pin1 knockdown blocks IRAK1 activation in human cells following TLR7 and TLR9, but not TLR3 stimulation. Human THP1 monocytes were infected with viral control shRNA or shRNA targeting Pin1 and simulated with poly (I:C) (TLR3), R-848 or CpG ligands for the indicated times, followed by analyzing the characteristic IRAK1 shift using immunoblotting. (d) In vivo kinase assay demonstrates IRAK1 kinase activity in Pin1 WT, but not Pin1 KO cells. Retroviral FLAG-IRAK1, and KD-IRAK1 or vector (VCT) control were coexpressed with a HA-N-terminal 220 aa fragment of IRAK1 as a substrate in Pin1 WT and KO MEFs (schematic diagram). IRAK1 kinase activity was determined by immunoblotting with HA antibodies to assess the characteristic mobility shift in IRAK1 N-terminal 220 aa due to trans-phosphorylation by co-expressed IRAK1 proteins. (e) Pin1 KO abolishes TLR dependent activation of exogenous IRAK1 in vivo . FLAG-IRAK1 and its KD mutant were co-expressed with TLR7 in Pin1 WT and KO MEF cells using retroviral vectors and stimulated with R-848 for the indicated times, followed by analyzing the characteristic IRAK1 mobility shift using immunoblotting. (f) Pin1, but not its WW domain-binding mutant (W34A) or catalytically inactive PPIase domain mutant (K63A), fully rescues IRAK1 activation in Pin1 KO cells. Pin1 KO MEFs stably expressing FLAG-IRAK1 were transfected with either WT-Pin1, K63A-Pin1, W34A-Pin1 or PPIase domain of Pin1 and TLR7 and stimulated for the indicated times, followed by analyzing the characteristic IRAK1 mobility shift using immunoblotting. Results are representative of at least three independent experiments.

Article Snippet: Phosphorylated Ser173-specific IRAK1 antibodies were raised by immunizing rabbits with a KLH-coupled phosphorylated Ser173-containing IRAK1 peptide (Proteintech Group) and were affinity purified, as described .

Techniques: Activation Assay, Derivative Assay, Expressing, Western Blot, Activity Assay, Knockdown, Infection, Control, shRNA, In Vivo, Kinase Assay, Retroviral, Plasmid Preparation, Mobility Shift, Phospho-proteomics, Mutagenesis, Binding Assay, Stable Transfection, Transfection

Journal: Nature immunology

Article Title: Essential role for the prolyl isomerase Pin1 in Toll-like receptor signaling and type I interferon-mediated immunity

doi: 10.1038/ni.2069

Figure Lengend Snippet: (a) Activated and phosphorylated IRAK1 is released from MyD88 in Pin1 WT cells, but inactive IRAK1 is not in Pin1 KO cells. HA-MyD88 and FLAG-IRAK1 were co-expressed in Pin1 WT and KO MEF using retroviral expression vectors, followed by immunoprecipitation with anti-HA antibody and then immunoblotting with anti-FLAG antibody. (b) Pin1 knockdown inhibits the interaction of IRF7 with TRAF6. THP1 cells expressing Pin1 -RNAi or control RNAi were stimulated with CpG for the indicated times and the interaction of IRF7 and TRAF6 was examined by Co-IP. (c, d) Pin1 knockdown prevents IRF7 nuclear translocation in human THP1 cells. Following TLR7 (c) or TLR9 (d) ligation for the indicated times, nuclear and cytoplasmic fractions of THP1 cells were prepared, followed by immunoblotting with IRF7 antibody. The purity of nuclear and cytosolic fractions was evaluated by immunoblotting with tubulin or lamin A/C antibodies, respectively. (e) Pin1 KO prevents IRF7 nuclear translocation after TLR7 or TLR9 ligation in pDCs. After R484 or CpG stimulation, Pin1 WT and KO pDCs were immunostained with IRF7 antibodies and counter-stained with DAPI, followed by confocal microscopy. Results are representative of at least three independent experiments.

Article Snippet: Phosphorylated Ser173-specific IRAK1 antibodies were raised by immunizing rabbits with a KLH-coupled phosphorylated Ser173-containing IRAK1 peptide (Proteintech Group) and were affinity purified, as described .

Techniques: Retroviral, Expressing, Immunoprecipitation, Western Blot, Knockdown, Control, Co-Immunoprecipitation Assay, Translocation Assay, Ligation, Staining, Confocal Microscopy

Journal: Nature immunology

Article Title: Essential role for the prolyl isomerase Pin1 in Toll-like receptor signaling and type I interferon-mediated immunity

doi: 10.1038/ni.2069

Figure Lengend Snippet: (a, b) Pin1 is required for IRF7 activation in response to TLR7 or TLR9 activation. Pin1 WT and KO cells transiently co-expressing a UAS (GAL) -reporter plasmid, Gal4-IRF7 and TLR7 (a) or TLR9 (b) were stimulated with R-848 or CpG, respectively, followed by luciferase assay 12 h later using renilla luciferase to normalize for transfection efficiency. (c, d) Re-expression of Pin1, but not its mutants, fully rescues impaired IRF7 activation and IFN-α production in Pin1 KO cells. Pin1 WT and KO MEFs stably expressing IRAK1 were transiently co-transfected with UAS (GAL) and Gal4-IRF7 and empty vector (EV), Pin1, WW domain mutant (W34A) or PPIase domain mutant (K63A), followed by luciferase assay (c) and IFN-α ELISA (d) , with Pin1 WT MEFs stably expressing IRAK1 transfected with EV as a control. Expression levels of WT, W34A and K63A Pin1 proteins are shown below graphs in (c) and (d) . (e) Overexpression of KD IRAK1 inhibits IRF7 activity in Pin1 WT, but does not affect basal IRF7 activity in Pin1 KO MEFs. Pin1 WT and KO MEFs were transiently transfected with Gal4-IRF7, UAS (Gal) , MyD88 (20 ng) and various amounts of KD Irak1 or control vector, as indicated, followed by assaying IRF7 activity using Renilla as a control for normalization. (f, g) Pin1 KO or IRAK1 mutations that prevent IRAK1 from being a Pin1 substrate abolish IRF7 activation and IFN-α production. Pin1 WT and KO cells stably expressing empty vector (EV), IRAK1 or IRAK1 mutants S110A, S131, S144, S173A, 3A (S131+S144+S173A) or KD were co-transfected with UAS (GAL) and Gal4-IRF7 to assess IRF7 reporter activity (f) or with IRF7 to measure IFN-α production (g) . Expression levels of IRAK1 and its various mutants are shown below the graph (f) . (h, i) Pin1 KO or Irak1 mutations that prevent IRAK1 from being a Pin1 substrate abolish antiviral activity. VSV production in plaque-forming units (PFU) per ml 24 h after infection of monolayer L cells (0.1 PFU/cell) previously treated with supernatants from Pin1 WT and KO cells stably expressing EV, IRAK1 or IRAK1 mutants S110A, S131, S144, S173A, 3A or KD (h) , with representative pictures of VSV plaques shown in (i) . ND, not detectable. Results shown are means ± s.d. of triplicates.

Article Snippet: Phosphorylated Ser173-specific IRAK1 antibodies were raised by immunizing rabbits with a KLH-coupled phosphorylated Ser173-containing IRAK1 peptide (Proteintech Group) and were affinity purified, as described .

Techniques: Activation Assay, Expressing, Plasmid Preparation, Luciferase, Transfection, Stable Transfection, Mutagenesis, Enzyme-linked Immunosorbent Assay, Control, Over Expression, Activity Assay, Infection

Journal: PLoS Pathogens

Article Title: HCV-Induced miR-21 Contributes to Evasion of Host Immune System by Targeting MyD88 and IRAK1

doi: 10.1371/journal.ppat.1003248

Figure Lengend Snippet: Huh7 hepatocytes were transfected with miR-21 mimics or control RNA, miR-21 inhibitor or control inhibitor (final concentration, 50 nM). After 48 h, MyD88, IRAK1, IRAK4, and TRAF6 mRNA levels were determined by qPCR ( A , B , C , and D ) and RT-PCR ( E and F ), respectively. Data are presented as the means SD (n = 3) from one representative experiment. Similar results were obtained in three independent experiments. **, p<0.01; *, p<0.05.

Article Snippet: Antibodies against HCV-core, STAT1, IRF-7, PKR, OAS, Mx, IFN-α/β Rα, IFN-α/β Rβ, phosphor-NF-κB p65, NF-κB p65, phosphor-ERK, ERK, phosphor-JNK, JNK, phosphor-c-Fos, c-Fos, phosphor-c-Jun, c-Jun, MyD88, IRAK1, and anti-mouse normal IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Transfection, Control, Concentration Assay, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS Pathogens

Article Title: HCV-Induced miR-21 Contributes to Evasion of Host Immune System by Targeting MyD88 and IRAK1

doi: 10.1371/journal.ppat.1003248

Figure Lengend Snippet: ( A ) Sequence alignment of miR-21 and its binding sites in the 3′ UTRs of MyD88 and IRAK1, as predicted by RNA22 software. ( B ) Huh7 hepatocytes (1×10 4 ) were co-transfected with pGL3-Basic, pGL3-MyD88 3′ UTR, or pGL3-IRAK1 3′ UTR firefly luciferase reporter plasmids (80 ng) and pRL-TK Renilla luciferase plasmid (40 ng), together with miR-21 mimics or control RNA, miR-21 inhibitor or control inhibitor (final concentration, 50 nM), as indicated. After 48 h, firefly luciferase activity was determined and normalized to Renilla luciferase activity. ( C ) HEK293 cells (1×10 4 ) were co-transfected with GFP control, GFP-MyD88 3′ UTR, or GFP-IRAK1 3′ UTR plasmid (400 ng), together with miR-21 mimics or control RNA (final concentration, 50 nM), as indicated. After 48 h, GFP expression was analyzed by FACS, and the mean fluorescence intensity (MFI) of GFP was determined. ( D and E ) Huh7 hepatocytes (1×10 6 ) were transfected with miR-21 mimics ( D ) or miR-21 inhibitor ( E ) at various concentrations for 48 h ( left ), or at 50 nM (final concentration) for the indicated time ( right ). MyD88 and IRAK1 protein levels were determined by Western blot and normalized to β-actin ( top panel ); MyD88 and IRAK1 mRNA levels were determined by qPCR and normalized to GAPDH ( bottom panel ). Data are presented as the means SD (n = 3) from one representative experiment. Similar results were obtained in three independent experiments. **, p<0.01; *, p<0.05.

Article Snippet: Antibodies against HCV-core, STAT1, IRF-7, PKR, OAS, Mx, IFN-α/β Rα, IFN-α/β Rβ, phosphor-NF-κB p65, NF-κB p65, phosphor-ERK, ERK, phosphor-JNK, JNK, phosphor-c-Fos, c-Fos, phosphor-c-Jun, c-Jun, MyD88, IRAK1, and anti-mouse normal IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Sequencing, Binding Assay, Software, Transfection, Luciferase, Plasmid Preparation, Control, Concentration Assay, Activity Assay, Expressing, Fluorescence, Western Blot

Journal: PLoS Pathogens

Article Title: HCV-Induced miR-21 Contributes to Evasion of Host Immune System by Targeting MyD88 and IRAK1

doi: 10.1371/journal.ppat.1003248

Figure Lengend Snippet: ( A ) Huh7 hepatocytes were transfected with nonspecific control siRNA or siRNA against MyD88 or IRAK1, as indicated. After 24 h, MyD88 and IRAK1 mRNA levels were determined by qPCR and normalized to GAPDH ( lower panel ); after 48 h, MyD88 and IRAK1 protein levels were determined by Western blot and normalized to β-actin ( upper panel ). ( B ) Huh7 hepatocytes were co-transfected with miR-21 mimic or control RNA and siRNA against MyD88 or IRAK1, as indicated. After 48 h, Huh7 cells were transfected with FL-J6/JFH5′C19Rluc2AUbi (0.1 µg) for the indicated time, and IFN-α expression and secretion were determined by qPCR and ELISA, respectively. ( C ) Huh7 hepatocytes were co-transfected with miR-21 inhibitor or control inhibitor and siRNA against MyD88 or IRAK1 or nonspecific control siRNA, as indicated. After 48 h, Huh7 cells were transfected with FL-J6/JFH5′C19Rluc2AUbi (0.1 µg) for the indicated times, and IFN-α expression and secretion were determined by qPCR and ELISA, respectively. Data are the means SD (n = 3) of one representative experiment. Similar results were obtained in three independent experiments. **, p<0.01; *, p<0.05.

Article Snippet: Antibodies against HCV-core, STAT1, IRF-7, PKR, OAS, Mx, IFN-α/β Rα, IFN-α/β Rβ, phosphor-NF-κB p65, NF-κB p65, phosphor-ERK, ERK, phosphor-JNK, JNK, phosphor-c-Fos, c-Fos, phosphor-c-Jun, c-Jun, MyD88, IRAK1, and anti-mouse normal IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

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

Journal: PLoS Pathogens

Article Title: HCV-Induced miR-21 Contributes to Evasion of Host Immune System by Targeting MyD88 and IRAK1

doi: 10.1371/journal.ppat.1003248

Figure Lengend Snippet: During HCV infection, the virus is first recognized by TLRs and RIG-1, which in turn activates MyD88 and IRAK1 to initiate IFN-α synthesis, resulting in the activation of ISGs and the inhibition of HCV replication. In addition, miR-21 expression is activated during HCV infection through two signaling pathways: the PKCε/JNK/c-Jun pathway and the PKCα/ERK/c-Fos pathway. The HCV NS5A protein activates PKCε to enhance the expression of JNK and c-Jun, while the HCV NS3/4A complex stimulates PKCα to promote the production of ERK and c-Fos. The two subunits (c-Jun and c-Fos) of AP-1 join together to recognize the miR-21 promoter and activate the expression of miR-21, which represses the expression of MyD88 and IRAK1 via imperfect base pairing between miR-21 and the 3′UTR of MyD88 and IRAK1. The reduction in MyD88 and IRAK1 causes a reduction of type-I IFN production and ISG expression that might contribute to viral pathogenesis and virus propagation.

Article Snippet: Antibodies against HCV-core, STAT1, IRF-7, PKR, OAS, Mx, IFN-α/β Rα, IFN-α/β Rβ, phosphor-NF-κB p65, NF-κB p65, phosphor-ERK, ERK, phosphor-JNK, JNK, phosphor-c-Fos, c-Fos, phosphor-c-Jun, c-Jun, MyD88, IRAK1, and anti-mouse normal IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Infection, Virus, Activation Assay, Inhibition, Expressing, Protein-Protein interactions

Journal: The Journal of Experimental Medicine

Article Title: Distinct Mutations in IRAK-4 Confer Hyporesponsiveness to Lipopolysaccharide and Interleukin-1 in a Patient with Recurrent Bacterial Infections

doi: 10.1084/jem.20030701

Figure Lengend Snippet: Identification and functionality of a point mutation in patient's IRAK-4 . (A) Illustration of C877T substitution mutation (M #1), resulting in a truncated form of IRAK-4. Arrow indicates approximate location of truncation in IRAK-4 protein. (B) Vectors encoding the WT (N) or C877T mutation (M #1) forms of IRAK-4 were expressed in HEK293T cells (5 μg vector/transfection) and cell lysates subjected to Western blot analysis using anti-Flag Ab. (C) Overexpression of WT (N) or C877T (M #1) forms of IRAK-4 in HEK293T cells failed to induce NF-κB–induced reporter activity. Cells were transiently transfected with pELAM-Luc, pCMV-βGal, and the indicated amounts of expression vectors encoding either IRAK-1 or normal (N) and mutated (M) forms of IRAK-4 (total amount of plasmid DNA was kept constant at 1.5 μg per transfection). After recovery for 48 h, NF-κB reporter activity was measured. The data represent the mean ± SEM of a representative experiment ( n = 3).

Article Snippet: Site-directed mutagenesis of WT human IRAK-1 and IRAK-4 expression vectors (pRK5-IRAK-1 and pRK7-IRAK-4; Tularik, Inc.) was performed using the QuickChange Site-Directed Mutagenesis Kit (Stratagene).

Techniques: Mutagenesis, Plasmid Preparation, Transfection, Western Blot, Over Expression, Activity Assay, Expressing

Journal: The Journal of Experimental Medicine

Article Title: Distinct Mutations in IRAK-4 Confer Hyporesponsiveness to Lipopolysaccharide and Interleukin-1 in a Patient with Recurrent Bacterial Infections

doi: 10.1084/jem.20030701

Figure Lengend Snippet: (A) Overexpression of WT (N), but not C877T mutant (M), IRAK-4 expression vector inhibits LPS- and IL-1–mediated signaling in HEK293T cells. Cells were transiently transfected as described in , permitted to recover for 48 h, and stimulated with medium, LPS, or rIL-1β for 5 h before measurement of NF-κB reporter activity. (B) Overexpression of WT (N), but not C877T mutant (M), IRAK-4 expression vectors differentially modulate IRAK-1 kinase activity. Cells were transiently transfected with control pCDNA3.1, pRK7-IRAK4 (N), or pRK7-IRAK4 (M; 5 μg per transfection). After 40 h recovery, cells were stimulated with rIL-1β for the indicated times, followed by lysis of cells and the IRAK-1 kinase assay ( n = 3).

Article Snippet: Site-directed mutagenesis of WT human IRAK-1 and IRAK-4 expression vectors (pRK5-IRAK-1 and pRK7-IRAK-4; Tularik, Inc.) was performed using the QuickChange Site-Directed Mutagenesis Kit (Stratagene).

Techniques: Over Expression, Mutagenesis, Expressing, Plasmid Preparation, Transfection, Activity Assay, Lysis, Kinase Assay

Journal: The Journal of Experimental Medicine

Article Title: Distinct Mutations in IRAK-4 Confer Hyporesponsiveness to Lipopolysaccharide and Interleukin-1 in a Patient with Recurrent Bacterial Infections

doi: 10.1084/jem.20030701

Figure Lengend Snippet: Identification and functionality of deletion mutation in patient's IRAK-4 . (A) Illustration of effect of AC deletion at nucleotides 620-621 in the patient (620-621del), resulting in a truncated form of IRAK-4 (M #2). Arrow indicates approximate location of truncation in IRAK-4 protein. (B) Vectors encoding the WT (N) or mutated (M #2) forms of IRAK-4 were expressed in HEK293T cells (10 μg vector/transfection), and cell lysates subjected to Western blot analysis using anti-Flag Ab as described in . (C) Overexpression of WT (N), but not the 620-621 deleted mutant (M #2), IRAK-4 expression vectors (10 μg/transfection) differentially modulate IRAK-1 kinase activity (see B; n = 3).

Article Snippet: Site-directed mutagenesis of WT human IRAK-1 and IRAK-4 expression vectors (pRK5-IRAK-1 and pRK7-IRAK-4; Tularik, Inc.) was performed using the QuickChange Site-Directed Mutagenesis Kit (Stratagene).

Techniques: Mutagenesis, Plasmid Preparation, Transfection, Western Blot, Over Expression, Expressing, Activity Assay

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: Anti-actin rabbit and mouse (4970 and 3700), MYC tag (2272), IRAK1 (4504), MYD88 (4283), NF-κB p65 (8242), and NF-κB Pp65 (3022) antibodies were purchased from Cell Signaling.

Techniques: Western Blot, Knock-Out, Control, 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: Anti-actin rabbit and mouse (4970 and 3700), MYC tag (2272), IRAK1 (4504), MYD88 (4283), NF-κB p65 (8242), and NF-κB Pp65 (3022) antibodies were purchased from Cell Signaling.

Techniques: Western Blot, Knock-Out, Control, 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 ΔIRAK1 cells. (A) Western blot for p-NF-κB and the IRAK pathway proteins in WT and ΔIRAK1 BCBL-1Cas9 cells 15 min post-IL-1β stimulation (1 ng/μl IL-1β). (B) Quantification of luciferase production using an NF-κB reporter assay system. Two ΔIRAK1 clones and WT BCBL-1Cas9 cells were stimulated with 1 ng/μl IL-1β or mock PBS. Cells were stimulated 24 h following transfection, and luciferase values were measured 6 h poststimulation. Results are fold change over mock. (C) Two ΔIRAK1 clones and WT BCBL-1Cas9 cells were stimulated with TNF-α, and the response was compared to that with mock using the same procedure as for panel B.

Article Snippet: Anti-actin rabbit and mouse (4970 and 3700), MYC tag (2272), IRAK1 (4504), MYD88 (4283), NF-κB p65 (8242), and NF-κB Pp65 (3022) antibodies were purchased from Cell Signaling.

Techniques: Activation Assay, Functional Assay, Western Blot, Luciferase, Reporter Assay, Clone Assay, 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: Anti-actin rabbit and mouse (4970 and 3700), MYC tag (2272), IRAK1 (4504), MYD88 (4283), NF-κB p65 (8242), and NF-κB Pp65 (3022) antibodies were purchased from Cell Signaling.

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: Anti-actin rabbit and mouse (4970 and 3700), MYC tag (2272), IRAK1 (4504), MYD88 (4283), NF-κB p65 (8242), and NF-κB Pp65 (3022) antibodies were purchased from Cell Signaling.

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: Anti-actin rabbit and mouse (4970 and 3700), MYC tag (2272), IRAK1 (4504), MYD88 (4283), NF-κB p65 (8242), and NF-κB Pp65 (3022) antibodies were purchased from Cell Signaling.

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