Journal: Cells

Article Title: The Expressions of TSLP, IL-33, and IL-17A in Monocyte Derived Dendritic Cells from Asthma and COPD Patients are Related to Epithelial–Macrophage Interactions

doi: 10.3390/cells9091944

Figure Lengend Snippet: TSLPR, ST2, IL17RA expression on moDCs upon co-cultivation with epithelium and monocyte derived macrophages (moMφs) in the multi-cell model culture of controls, patients with asthma and patients with COPD.

Article Snippet: Cells were stained with antibodies against the surface binding molecules: TSLPR BV421 (clone 1F11/TSLPR), CD45 APC-H7 (clone 2D1), CD14 BV605 (clone M5E2), CD83 BV510 (clone HB15e), CD11B APC-R700 (Clone M1/70) (BD Biosciences, San Jose, CA, USA) ST2 APC (Biotechne, R&D Systems, Minneapolis, MN, USA), IL17RA PE (clone REA290) (Miltenyi Biotec, Bergisch Gladbach, Germany) in BD Horizon Brilliant Stain Buffer (BD Biosciences) and incubated for 20 min in the dark at room temperature.

Techniques: Expressing, Derivative Assay

Journal: iScience

Article Title: Pharmacological inhibition of RAS overcomes FLT3 inhibitor resistance in FLT3-ITD+ AML through AP-1 and RUNX1

doi: 10.1016/j.isci.2024.109576

Figure Lengend Snippet:

Article Snippet: CD217 (IL17RA) Antibody, anti-human, APC, REAfinity , Miltenyi Biotec , Cat# 130-127-293; RRID: AB_2904838.

Techniques: Control, Virus, Recombinant, Modification, Saline, Stripping Membranes, Protease Inhibitor, Reverse Transcription, Membrane, Labeling, Gel Extraction, Plasmid Preparation, RNA Library Preparation, Library Quantification, Software

Journal: Cell Death & Disease

Article Title: Cardiomyocyte IL-1R2 protects heart from ischemia/reperfusion injury by attenuating IL-17RA-mediated cardiomyocyte apoptosis

doi: 10.1038/s41419-022-04533-1

Figure Lengend Snippet: A Relative FPKM levels of chemokine genes related to IL-17A signaling are shown ( n = 3). The differentially expressed mRNAs and genes were selected with log2 (fold change) > 1 or log2 (fold change) < −1 and with statistical significance ( p -value < 0.05). B, C Representative western blots and quantification of IL-17RA expression in the heart from IL-1R2+/+ and IL-1R2−/− mice subjected to myocardial I/R injury ( n = 3 per group). D, F Representative western blots and quantification of IL-17RA expression in NRVMs transfected with siRNA to block IL-1R2 expression, followed by IL-1β treatment ( n = 3 per group). E, G–I Representative western blots and quantification of STAT-1, p-STAT-1, Bcl-xL, and Bax expression in NRVMs with or without a plasmid transfection for overexpression of IL-1R2 followed by IL-1β and IL-17A treatment ( n = 5 per group). * P < 0.05 and ** P < 0.01. Two-tailed unpaired Student’s t -test for two groups or one-way ANOVA for three groups or more.

Article Snippet: After blocking with 5% skimmed milk for 1 h, the membranes were incubated with antibodies against IL-1R1 (1:1000, Santa Cruz, Biotechnology, Santa Clara, CA, USA), IL-1R2 (1:1000, Novus Biologicals, Littleton, CO, USA), p-NF-κB (1:1000, CST), JAK2 (1:1000, CST), p-JAK2 (1:1000, CST), STAT1 (1:1000, CST), p-STAT1 (1:1000, CST), STAT3 (1:1000, CST), p-STAT3 (1:1000, CST), Bcl-xL (1:1000, CST), Bax (1:1000, Proteintech, Rosemont, IL, USA), IL-17RA (1:1000, Abclonal, Wuhan, China), nitrotyrosine (1:1000, Sigma-Aldrich) and GAPDH (1:5000, CST) overnight at 4 °C.

Techniques: Western Blot, Expressing, Transfection, Blocking Assay, Plasmid Preparation, Over Expression, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Cardiomyocyte IL-1R2 protects heart from ischemia/reperfusion injury by attenuating IL-17RA-mediated cardiomyocyte apoptosis

doi: 10.1038/s41419-022-04533-1

Figure Lengend Snippet: A, B Representative images and western blots for confirming the expression of IL-1R2 in cardiomyocytes with AAV9-GFP and AAV9-IL-1R2 injection. C–E Representative echocardiographic images and quantification of sham mice ( n = 7), GFP ( n = 7) and IL-1R2-overexpression mice ( n = 7) subjected to myocardial I/R surgery. F–H Representative images of 2,3,5-triphenyltetrazolium chloride staining on mice subjected to myocardial I/R surgery and quantification of the relative ratios of infarct area to area at risk (border zone) as well as the relative ratios of the area at risk to left ventricle area in the GFP ( n = 5) and IL-1R2–overexpressing ( n = 5) groups; Scale bar: 1 mm. I Quantitation of TUNEL staining on the heart sections from the Sham ( n = 3), GFP ( n = 5), and IL-1R2–overexpressing ( n = 5) mice subjected to myocardial I/R surgery. Scale bar: 100 µm. J, K Representative western blots and quantification of Stat1, p-Stat1, Bcl-xL, Bax, and IL-17RA expression in the hearts from Sham, GFP, and IL-1R2–overexpressing mice subjected to myocardial I/R injury. Scale bar: 100 µm. * P < 0.05 and ** P < 0.01. Two-tailed unpaired Student’s t -test for two groups or one-way ANOVA for three groups or more.

Article Snippet: After blocking with 5% skimmed milk for 1 h, the membranes were incubated with antibodies against IL-1R1 (1:1000, Santa Cruz, Biotechnology, Santa Clara, CA, USA), IL-1R2 (1:1000, Novus Biologicals, Littleton, CO, USA), p-NF-κB (1:1000, CST), JAK2 (1:1000, CST), p-JAK2 (1:1000, CST), STAT1 (1:1000, CST), p-STAT1 (1:1000, CST), STAT3 (1:1000, CST), p-STAT3 (1:1000, CST), Bcl-xL (1:1000, CST), Bax (1:1000, Proteintech, Rosemont, IL, USA), IL-17RA (1:1000, Abclonal, Wuhan, China), nitrotyrosine (1:1000, Sigma-Aldrich) and GAPDH (1:5000, CST) overnight at 4 °C.

Techniques: Western Blot, Expressing, Injection, Over Expression, Staining, Quantitation Assay, TUNEL Assay, Two Tailed Test

Journal:

Article Title: Cutting Edge: Identification of a Pre-Ligand Assembly Domain (PLAD) and Ligand Binding Site in the IL-17 Receptor 1

doi:

Figure Lengend Snippet: The FN2 domain of IL-17RA mediates ligand-independent assembly. A, Predicted structure of IL-17RA FN domains. PHYRE predicted two FN domains within the mouse IL-17RA ECD. Yellow, β-Sheets; red, α-helices; green, unstructured loops; blue, turns. Sequences of each domain are shown. B, Schematic diagram of IL-17RA FRET and Y2H constructs. SEFIR is a major signaling domain in IL-17RA (22). AD, Activation domain; BD, binding domain. C, FN2linker drives ligand-independent association in cells. HEK293 cells expressing the indicated combinations of IL-17RA constructs fused to CFP or YFP were assayed for N-FRET in the absence (open bars) or presence of IL-17 (filled bars) or IL-17F (gray bar) for 10 min. Significance was assessed by t test, p < 0.05; n.s., not significant. D, Representative images of IL-17RAΔ/CFP plus IL-17RAΔFN2linker/YFP. CFP, YFP, and FRET emissions are shown. Unstim., Unstimulated. E, IL-17RA can co-IP with IL-17RAΔFN2linker. HEK293 cells expressing IL-17RAΔFN2linker/YFP were transiently transfected with IL-17RA.HA. Cells were lysed and immunoprecipitated (IP) with anti-HA or M177 anti-IL-17RA Abs and immunoblotted with Abs to GFP (which cross-react with YFP) or HA. Note that we always observe slight cross-reactivity of anti-HA Abs with YFP and CFP for unknown reasons. W, Western blot.

Article Snippet: Flow cytometry and fluorescence resonance energy transfer (FRET) Cells were stained with M750 or M177 anti-IL-17RA and anti-rat PE (BD Pharmingen).

Techniques: Construct, Activation Assay, Binding Assay, Expressing, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Western Blot

Journal:

Article Title: Cutting Edge: Identification of a Pre-Ligand Assembly Domain (PLAD) and Ligand Binding Site in the IL-17 Receptor 1

doi:

Figure Lengend Snippet: Homotypic interactions between IL-17RA occur via the FN2 domain a

Article Snippet: Flow cytometry and fluorescence resonance energy transfer (FRET) Cells were stained with M750 or M177 anti-IL-17RA and anti-rat PE (BD Pharmingen).

Techniques: Construct

Journal:

Article Title: Cutting Edge: Identification of a Pre-Ligand Assembly Domain (PLAD) and Ligand Binding Site in the IL-17 Receptor 1

doi:

Figure Lengend Snippet: Model of IL-17RA subunit dynamics. Data suggest that the cytoplasmic tails of IL-17RA are held in proximity before ligand binding (A), but separate upon binding IL-17 (B). Based on FRET, at least one FN1 domain is sufficient to mediate ligand-induced subunit reconfiguration (C). However, in the absence of both FN1 domains the cytoplasmic tails show increased association (D). IL-17 requires the FN2 and linker regions to bind the receptor but may also contact FN1 (B–D).

Article Snippet: Flow cytometry and fluorescence resonance energy transfer (FRET) Cells were stained with M750 or M177 anti-IL-17RA and anti-rat PE (BD Pharmingen).

Techniques: Ligand Binding Assay, Binding Assay

Journal:

Article Title: Cutting Edge: Identification of a Pre-Ligand Assembly Domain (PLAD) and Ligand Binding Site in the IL-17 Receptor 1

doi:

Figure Lengend Snippet: Requirements for IL-17 signaling and binding. A, The FN1 domain is dispensable but the linker is required for IL-17-dependent signaling. IL-17RA−/−fibroblasts were transfected in triplicate with the indicated IL-17RA constructs (with FL cytoplasmic tails) and 24p3-Luc (18). Cells were stimulated with IL-17 (200 ng/ml) and/or TNF-α (2 ng/ml). After 6 h, luciferase activity was determined and normalized to Renilla luciferase. B and C, IL-17RA-neutralizing Abs bind the FN2linker domain. B, HEK293 cells stably transfected with IL-17RΔ/CFP (top) or transiently transfected with IL-17RAΔFN2linker/CFP (bottom) were incubated with a non-neutralizing (clone M177) or neutralizing Ab (clone M750) to murine IL-17RA. Filled histograms are isotype controls. C, Whole cell lysates from HEK293 cells stably expressing IL-17RAΔ/CFP or IL-17RAΔFN2linker/YFP were immunoprecipitated (IP) with M750 or M177 as indicated and blotted with anti-GFP Abs. D and E, FN2linker has a reduced affinity for IL-17. HEK293 cells stably expressing IL-17RAΔ/CFP or IL-17RAΔFN2linker/YFP were stained with various concentrations of IL17.Fc after blocking endogenous human IL-17RA (D) or with M750 (E). F, The linker is required but not sufficient for IL-17 binding. HEK293 cells transiently transfected with IL-17RAΔ/YFP, IL-17RAΔFN1linker/YFP, or IL-17RAΔFN2/YFP were stained with IL17.Fc and gated on YFP-positive cells.

Article Snippet: Flow cytometry and fluorescence resonance energy transfer (FRET) Cells were stained with M750 or M177 anti-IL-17RA and anti-rat PE (BD Pharmingen).

Techniques: Binding Assay, Transfection, Construct, Luciferase, Activity Assay, Stable Transfection, Incubation, Expressing, Immunoprecipitation, Staining, Blocking Assay

Journal: Oncotarget

Article Title: Hyperinsulinemia enhances interleukin-17-induced inflammation to promote prostate cancer development in obese mice through inhibiting glycogen synthase kinase 3-mediated phosphorylation and degradation of interleukin-17 receptor

doi: 10.18632/oncotarget.7296

Figure Lengend Snippet: ( A – D ) 293 cells were co-transfected with Flag-IL-17RA, V5-IL-17RC, and HA-Act1, labeled with 32 P orthophosphate, and treated with 20 ng/ml IL-17A for 20 min; IP with anti-HA; autoradiography (A) followed by IB (B-D). ( E ) Anti-Flag IP followed by IB. ( F – G ) Co-IP of Flag-IL-17RA and HA-GSK3β. ( H ) 293 cells were co-transfected with Flag-IL-17RA, WT HA-GSK3β or kinase-dead mutant HA-GSK3βK85A, labeled with 32 P orthophosphate, or treated with 20 mM LiCl for 2 h; autoradiography ( 32 P) followed by IB. WE, whole cell extract.

Article Snippet: The antibodies and peptides used are as follows: rabbit anti-human phospho-IL-17RA (T780) polyclonal antibodies (B1, B2, B3, and B4), phosphorylated antigen peptide (C-LTDPH(pT)PYEEEQ) and non-phosphorylated antigen peptide (C-LTDPHTPYEEEQ) were produced through a contract by AbMART (Shanghai) CO., LTD., Shanghai, China.

Techniques: Transfection, Labeling, Autoradiography, Co-Immunoprecipitation Assay, Mutagenesis

Journal: Oncotarget

Article Title: Hyperinsulinemia enhances interleukin-17-induced inflammation to promote prostate cancer development in obese mice through inhibiting glycogen synthase kinase 3-mediated phosphorylation and degradation of interleukin-17 receptor

doi: 10.18632/oncotarget.7296

Figure Lengend Snippet: ( A – B ) 293 cells were transfected with Flag-IL-17RA and its mutants and labeled with 32 P orthophosphate; autoradiography ( 32 P) followed by IB. ( C ) IL-17RA peptide phosphorylated at T780 (p-Peptide) was treated with CIP, while IL-17RA peptide with wild-type T780 (Peptide) or with mutant T780A (mut-Peptide) were treated with recombinant GSK3, followed with dot blot analysis using B4 antibodies. ( D – E ) Flag-IL-17RA, Flag-IL-17RAT780A mutant, or empty vector was transfected into 293 cells; HeLa cells were not transfected; IP with anti-Flag, B4 (+), or control IgG (−), followed by IB; *indicates endogenous P-IL-17RA.

Article Snippet: The antibodies and peptides used are as follows: rabbit anti-human phospho-IL-17RA (T780) polyclonal antibodies (B1, B2, B3, and B4), phosphorylated antigen peptide (C-LTDPH(pT)PYEEEQ) and non-phosphorylated antigen peptide (C-LTDPHTPYEEEQ) were produced through a contract by AbMART (Shanghai) CO., LTD., Shanghai, China.

Techniques: Transfection, Labeling, Autoradiography, Mutagenesis, Recombinant, Dot Blot, Plasmid Preparation, Control

Journal: Oncotarget

Article Title: Hyperinsulinemia enhances interleukin-17-induced inflammation to promote prostate cancer development in obese mice through inhibiting glycogen synthase kinase 3-mediated phosphorylation and degradation of interleukin-17 receptor

doi: 10.18632/oncotarget.7296

Figure Lengend Snippet: ( A ) and ( D ) 239 cells stably expressing Flag-IL-17RA (293-IL-17RA cell line) and HeLa cells were treated with 2 μM AZD5363 (a pan-Akt inhibitor) and/or 50 ng/ml insulin for the indicated time periods; Western blot analysis was performed for the indicated proteins; exogenous, IL-17RA transfected into 293 cells; endogenous, endogenous IL-17RA expressed in 293 and HeLa cells. ( B ) and ( C ) 293-IL-17RA cells were treated with 20 ng/ml IL-17A, with or without 2 μM AZD5363 and/or 50 ng/ml insulin for 2 h; IL-17-downstream gene expression was determined using qRT-PCR analysis; * P < 0.05 compared to each single treatment group; ** P < 0.05 compared to the combined insulin and IL-17 treatment group (one-way ANOVA). ( E ) and ( F ) HeLa cells were treated with 20 ng/ml IL-17A, with or without 2 μM AZD5363 and/or 50 ng/ml insulin for 2 h; IL-17-downstream gene expression was determined using qRT-PCR analysis; * P < 0.05 compared to each single treatment group; ** P < 0.05 compared to the combined insulin and IL-17 treatment group (one-way ANOVA). Data represent the mean ± standard deviation of three independent experiments ( n = 3).

Article Snippet: The antibodies and peptides used are as follows: rabbit anti-human phospho-IL-17RA (T780) polyclonal antibodies (B1, B2, B3, and B4), phosphorylated antigen peptide (C-LTDPH(pT)PYEEEQ) and non-phosphorylated antigen peptide (C-LTDPHTPYEEEQ) were produced through a contract by AbMART (Shanghai) CO., LTD., Shanghai, China.

Techniques: Stable Transfection, Expressing, Western Blot, Transfection, Gene Expression, Quantitative RT-PCR, Standard Deviation

Journal: Oncotarget

Article Title: Hyperinsulinemia enhances interleukin-17-induced inflammation to promote prostate cancer development in obese mice through inhibiting glycogen synthase kinase 3-mediated phosphorylation and degradation of interleukin-17 receptor

doi: 10.18632/oncotarget.7296

Figure Lengend Snippet: ( A – D ) 293 cells stably expressing Flag-IL-17RA were treated with 20 mM LiCl or 10 μM MG132, and/or 50 μg/ml CHX; P-IL-17RA was detected using B4 and total exogenous IL-17RA was detected using anti-Flag; the levels of total exogenous IL-17RA (C) were normalized by the levels of GAPDH (D); exo, exogenously transfected IL-17RA; endo, endogenously expressed IL-17RA; * P < 0.05 compared to LiCl and MG132 treated groups (Student's t test). ( E ) Flag-IL-17RA and Flag-IL-17RA-T780A mutant were co-transfected with HA-tagged ubiquitin (HA-Ub), with or without 10 μM MG132 treatment; non-specific, an unknown band detected by anti-HA antibodies, which was not IL-17RA or IgG heavy chain (see ). ( F ) Flag-IL-17RA and Flag-IL-17RA-T780A mutant were co-transfected with HA-tagged ubiquitin (HA-Ub) or K48-only ubiquitin (HA-K48), with 10 μM MG132 treatment, followed by IP and IB.

Article Snippet: The antibodies and peptides used are as follows: rabbit anti-human phospho-IL-17RA (T780) polyclonal antibodies (B1, B2, B3, and B4), phosphorylated antigen peptide (C-LTDPH(pT)PYEEEQ) and non-phosphorylated antigen peptide (C-LTDPHTPYEEEQ) were produced through a contract by AbMART (Shanghai) CO., LTD., Shanghai, China.

Techniques: Stable Transfection, Expressing, Transfection, Mutagenesis, Ubiquitin Proteomics

Journal: Oncotarget

Article Title: Hyperinsulinemia enhances interleukin-17-induced inflammation to promote prostate cancer development in obese mice through inhibiting glycogen synthase kinase 3-mediated phosphorylation and degradation of interleukin-17 receptor

doi: 10.18632/oncotarget.7296

Figure Lengend Snippet: ( A – I ) Human normal prostate, PIN, and prostate cancer tissues were double stained for P-IL-17RA using B4 (arrowheads) and for basal cells using anti-p63 (arrows). ( J ) Quantification of P-IL-17RA staining.

Article Snippet: The antibodies and peptides used are as follows: rabbit anti-human phospho-IL-17RA (T780) polyclonal antibodies (B1, B2, B3, and B4), phosphorylated antigen peptide (C-LTDPH(pT)PYEEEQ) and non-phosphorylated antigen peptide (C-LTDPHTPYEEEQ) were produced through a contract by AbMART (Shanghai) CO., LTD., Shanghai, China.

Techniques: Staining