Journal: Nature Communications

Article Title: IL-13Rα2 uses TMEM219 in chitinase 3-like-1-induced signalling and effector responses

doi: 10.1038/ncomms12752

Figure Lengend Snippet: ( a ) Y2H demonstration of the interaction between IL-13Rα2 and TMEM219. ( b ) 1HAEo cells were transfected with hIL-13Rα2 (IL-13Rα2) and/or human TMEM219 (TMEM219) expressing plasmids. Co-IP performed with either anti-IL-13Rα2 (1:500, AF146, R&D) or anti-TMEM219 (1:500, sc-244405, Santa Cruz) antibody, and the precipitates were evaluated using IB analysis as noted. ( c ) Direct interaction between TMEM219 and IL-13Rα1 was tested by Co-IP/IB assay using recombinant human (rh) TMEM219 (350 ng), IL-13 (300 ng, 213-ILB, R&D) and IL-13Rα1 (500 ng, 146-IR, R&D). His-tagged human TMEM219 was expressed and purified in HEK 293T cells using pcDNA 3.1 vector. Commercially available Recombinant human IL-13 and IL-13Rα1 (R&D systems) were used for this assay. ( d ) Interaction of IL-13Rα2 and TMEM219 on cell membrane was confirmed by BiFC assay. IL-13Rα2 and TMEM219 constructs were generated which contain fragments of the tagging protein Venus YFP fragments (V1 and V2). A similar approach was used to generate the negative control, CCR3 containing Venus YFP fragments. The indicated plasmids were transfected into 1HAEo cells, which were treated with rChi3l1 (500 ng ml −1 , 2599-CH, R&D), rIL-13 (20 ng ml −1 , 213-ILB, R&D) or vehicle control as noted. ( e ) Immunohistochemical demonstration of the co-localization of IL-13Rα2 and TMEM219 in lungs from IL-13 Tg mice. Fluorescence images were counterstained with 4′6-diamidino-2-phenylindole (DAPI) for nucleus identification. ( f ) Y2H characterization of the IL-13Rα2 sequences critical for binding to hTMEM219. The extracellular domain (ECD), transmembrane domain (TD), intracellular domain (ICD), signal peptide (SP) and sites of N-glycosylation (N) are illustrated. Fragment binding is illustrated with a + sign. ( b , c ) Representative of two separate western blot analysis. ( d , e ) Composite illustration taken from the noted four groups of mice ( N >5) that behaved similarly.

Article Snippet: Tissue sections were then blocked with a non-serum protein-blocking reagent (DakoCytomation Inc., Mississauga, ON, Canada) for 15 min at room temperature and incubated with primary antibodies (anti-IL-13Rα2 (1/50 dilution, AF539, R&D systems), anti-TMEM219 (1/50 dilution, sc-244404, Santa Cruz Biotechnology Inc.) for 60 min at room temperature in a humid chamber.

Techniques: Transfection, Expressing, Co-Immunoprecipitation Assay, Recombinant, Purification, Plasmid Preparation, Membrane, Bimolecular Fluorescence Complementation Assay, Construct, Generated, Negative Control, Immunohistochemical staining, Fluorescence, Binding Assay, Western Blot

Journal: Nature Communications

Article Title: IL-13Rα2 uses TMEM219 in chitinase 3-like-1-induced signalling and effector responses

doi: 10.1038/ncomms12752

Figure Lengend Snippet: ( a , b ) Serial dilution of TMEM219 nanodisc (10–10,000 nM) were incubated with 10 nM of BIOPYD-labelled Chi3l1 or IL-13Rα2. No significant increase of fluorescence signals was noted in these nanodisc incubations. ( c ) Serial dilution of TMEM219 nanodisc (10–10,000 nM) were incubated with 10 nM of BIOPYD-labelled IL-13Rα2-Chi3l1 complexes. As the concentration of the nanodisc increases, changes in fluorescence signal increases until it reaches saturation. IL-13Rα2-Chi3l1 complexes directly bind to TMEM219 nanodisc with an affinity of 288 nM, respectively. All the panels are representative of a minimum of two separate experiments.

Article Snippet: Tissue sections were then blocked with a non-serum protein-blocking reagent (DakoCytomation Inc., Mississauga, ON, Canada) for 15 min at room temperature and incubated with primary antibodies (anti-IL-13Rα2 (1/50 dilution, AF539, R&D systems), anti-TMEM219 (1/50 dilution, sc-244404, Santa Cruz Biotechnology Inc.) for 60 min at room temperature in a humid chamber.

Techniques: Serial Dilution, Incubation, Fluorescence, Concentration Assay

Journal: Nature Communications

Article Title: IL-13Rα2 uses TMEM219 in chitinase 3-like-1-induced signalling and effector responses

doi: 10.1038/ncomms12752

Figure Lengend Snippet: ( a , b ) HB-EGF production by 1HAEo cells. 1HAEo cells were treated rhChi3l1 (500 ng ml −1 ) with (+) and without (−) siRNA silencing of IL-13Rα2 or IL-13Rα1 or TMEM219. The concentrations of HB-EGF were assessed by ELISA. ( c ) HB-EGF production by macrophages. Peritoneal macrophages were prepared from WT, IL-13Rα2 or TMEM219 null mutant mice and incubated with rmChi3l1 (500 ng ml −1 ) for 48 h. The levels of supernatant HB-EGF were assessed by ELISA. ( d ) Effects of anti-TMEM219 on HB-EGF production by 1HAEo cells. 1HAEo cells were stimulated for 48 h with rhChi3l1 (500 ng ml −1 ) in the presence and absence of TMEM219 neutralizing antibodies. In all panels, the levels of supernatant HB-EGF were assessed by ELISA. The values represent the mean±s.e.m. of triplicate evaluations in a minimum of three separate experiments. ** P <0.01; *** P <0.001; ns, non-significant by Student's t -test.

Article Snippet: Tissue sections were then blocked with a non-serum protein-blocking reagent (DakoCytomation Inc., Mississauga, ON, Canada) for 15 min at room temperature and incubated with primary antibodies (anti-IL-13Rα2 (1/50 dilution, AF539, R&D systems), anti-TMEM219 (1/50 dilution, sc-244404, Santa Cruz Biotechnology Inc.) for 60 min at room temperature in a humid chamber.

Techniques: Enzyme-linked Immunosorbent Assay, Mutagenesis, Incubation

Journal: Nature Communications

Article Title: IL-13Rα2 uses TMEM219 in chitinase 3-like-1-induced signalling and effector responses

doi: 10.1038/ncomms12752

Figure Lengend Snippet: ( a ) 1HAEo cells were stimulated with rhChi3l1 (500 ng ml −1 ) with (+) and without (−) the siRNA silencing of IL-13Rα2 or TMEM219 and western blot evaluations were undertaken to evaluate the activation of the MAPK/ERK and PKB/AKT pathways. ( b ) Peritoneal macrophages from WT, IL-13Rα2 null or TMEM219 null mutant mice were stimulated with rChi3l1 (500 ng ml −1 , 2649-CH, R&D) for 24 h and western blot evaluations were used to characterize the activation of the MAPK/ERK and PKB/AKT signalling pathways. ( c , d ) 1HAEo cells were stimulated with rhChi3l1 (500 ng ml −1 , R&D) with (+) and without (−) siRNA silencing of IL-13Rα1, IL-13Rα2 or TMEM219 and western blot evaluations of MAPK/ERK activation and β-catenin phosphorylation were undertaken as noted. ( e ) Peritoneal macrophages were isolated from IL-13Rα2 or TMEM219 null mutant mice, stimulated with rmChi3l1 (500 ng ml −1 , R&D) and western blot evaluations of β-catenin phosphorylation were undertaken. All the panels are representatives of a minimum of three separate experiments.

Article Snippet: Tissue sections were then blocked with a non-serum protein-blocking reagent (DakoCytomation Inc., Mississauga, ON, Canada) for 15 min at room temperature and incubated with primary antibodies (anti-IL-13Rα2 (1/50 dilution, AF539, R&D systems), anti-TMEM219 (1/50 dilution, sc-244404, Santa Cruz Biotechnology Inc.) for 60 min at room temperature in a humid chamber.

Techniques: Western Blot, Activation Assay, Mutagenesis, Isolation

Journal: Nature Communications

Article Title: IL-13Rα2 uses TMEM219 in chitinase 3-like-1-induced signalling and effector responses

doi: 10.1038/ncomms12752

Figure Lengend Snippet: ( a ) 1 HAEo cells were incubated with hydrogen peroxide (200 μM) or vehicle control for 24 h and cellular apoptosis was evaluated by flow cytometry using Annein-V and PI staining. These experiments were undertaken using 1HAEo cells that had been treated with siRNA that silenced IL-13Rα2 or TMEM219 or appropriate controls ( b ) WT and TMEM219 −/− macrophages were incubated in the presence and absence of rhChi3l1. In selected experiments, TMEM219 or an appropriate control was transfected and over expressed (TMEM219+). After 4 h of incubation, the percentage of TUNEL positive cells in three microscopic fields was assessed (× 10). ( c ) WT, IL-13Rα2 −/− and TMEM219 −/− mice were exposed to 100% oxygen for 72 h, their lungs were collected and cell death was evaluated using TUNEL staining. Representative microscopic images are illustrated. The arrows highlight selected TUNEL positive cells. ( d ) WT, IL-13Rα2 −/− and TMEM219 −/− mice were exposed to 100% oxygen for 72 h, their lungs were collected and cell death was evaluated using TUNEL staining. The TUNEL positive cells in the airways and parenchymal areas were quantitated by counting at least 10 microscopic fields under × 20 magnification. ( e ) WT, IL-13Rα2 −/− and TMEM219 −/− mice were exposed to 100% oxygen for 72 h. As an index of lung injury, the levels of total protein in BAL were quantitated. ( f ) Survival analysis of WT ( n =5), IL-13Rα2 −/− ( n =7) and TMEM219 −/− ( n =7) mice after exposure of 100% O 2 . The TMEM219 −/− and IL-13Rα2 −/− mice all had significantly decreased survival compared with WT mice ( P <0.01, log-rank Mantel–Haenszel test). ( a ) Representative of three separate experiments. ( c ) Composite illustration taken from the noted three groups of mice ( N ≥5) that behaved similarly. The values in b represent the mean±s.e.m. of triplicate evaluations in a minimum of three separate experiments. The values in d , e represent the mean±s.e.m. of evaluations in a minimum of five mice ** P <0.01; *** P <0.001. NS, not significant by Student's t -test.

Article Snippet: Tissue sections were then blocked with a non-serum protein-blocking reagent (DakoCytomation Inc., Mississauga, ON, Canada) for 15 min at room temperature and incubated with primary antibodies (anti-IL-13Rα2 (1/50 dilution, AF539, R&D systems), anti-TMEM219 (1/50 dilution, sc-244404, Santa Cruz Biotechnology Inc.) for 60 min at room temperature in a humid chamber.

Techniques: Incubation, Flow Cytometry, Staining, Transfection, TUNEL Assay