mouse anti hil 2  (R&D Systems)

Journal: OncoTargets and therapy

Article Title: Enhancement of antitumor activity by using a fully human gene encoding a single-chain fragmented antibody specific for carcinoembryonic antigen

doi: 10.2147/OTT.S140174

Figure Lengend Snippet: The scFv-IL2 fusion protein maintains the functions of both component proteins. Notes: ( A ) Schematic representation of the scFv-IL2 gene within the pBAD/gIII expression vector. The 45κHscFv and hIL-2 genes were combined by SOE-PCR. The scFv-IL2 gene was inserted into an Escherichia coli . expression vector. ( B ) Detection of scFv-IL2 using CBB staining and Western blotting. The scFv-IL2 and the parental 45κHscFv vectors were transformed into TOP10 cells and were expressed by the addition of D-arabinose. After concentration through an Ni + column, the proteins were detected by CBB staining (lanes 1 and 3) and Western blotting using specific antibodies (lanes 1 and 3, anti-His; lane 5, anti-hIL-2; lane 6, anti-c-myc antibodies), at the expected sizes (28 and 45 kbp, respectively). ( C ) scFv-IL2 was detected by sandwich ELISA using rabbit and mouse anti-hIL-2 antibodies. Various concentrations of scFv-IL2 were incubated in rabbit anti-hIL-2 antibody-coated 96-well microtiter plates, and were then incubated with the mouse anti-hIL-2 antibody, followed by an HRP-conjugated goat anti-mouse antibody. After reacting with the OPD substrate, the absorption at 490 nm was determined using a plate reader. The absorption of scFv-IL2 wells increased in a dose-dependent manner. ( D ) The biological function of IL-2 in the scFv-IL2 fusion protein was determined using a cell proliferation assay. CTLL-2 cells were incubated with various concentrations of IL-2 and the equivalent scFv-IL2. After 24 h incubation, the cells were detected by WST-8 assay. CTLL-2 cells were proliferated by adding scFv-IL2 and IL-2. ( E and F ) The biological function of scFv antibody in the scFv-IL2 treated cells was detected by flow cytometry. Schematic representation of functional analysis of scFv antibody in scFv-IL2-treated cells. scFv-IL2 creates a bridge between MKN-45 CEA-positive cells and FITC-labeled anti-hIL-2 antibody ( E ). The fluorescence intensity of FITC was shifted to the right in the presence of scFv-IL2, compared with IL-2 ( F ). Abbreviations: CBB, Coomassie Brilliant Blue; CEA, carcinoembryonic antigen; FITC, fluorescein isothiocyanate; OPD, o-Phenylenediamine-2HCl; scFv, single-chain fragmented antibody; SOE, splice-overlap extension.

Article Snippet: Mouse anti-His and anti-hIL-2 antibodies and rabbit anti-c-myc and anti-hIL-2 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Expressing, Plasmid Preparation, Overlap Extension Polymerase Chain Reaction, Staining, Western Blot, Transformation Assay, Concentration Assay, Sandwich ELISA, Incubation, Proliferation Assay, Flow Cytometry, Functional Assay, Labeling, Fluorescence

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: Lysates from these cells were subjected to immunoprecipitation using anti-hIL-13Rα2 mouse monoclonal antibody (1/500 dilution, AF146, R&D system, Minneapolis, USA) or anti-TMEM219 polyclonal antibody (1/500 dilution, sc-244404, Santa Cruz Biotechnology Inc., CA, USA).

Techniques: Transfection, Expressing, Co-Immunoprecipitation Assay, Recombinant, Purification, Plasmid Preparation, Membrane, Bimolecular Fluorescence Complementation Assay, Construct, Generated, Negative Control, Control, Immunohistochemical staining, Fluorescence, Binding Assay, Glycoproteomics, 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: Lysates from these cells were subjected to immunoprecipitation using anti-hIL-13Rα2 mouse monoclonal antibody (1/500 dilution, AF146, R&D system, Minneapolis, USA) or anti-TMEM219 polyclonal antibody (1/500 dilution, sc-244404, Santa Cruz Biotechnology Inc., CA, USA).

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: Lysates from these cells were subjected to immunoprecipitation using anti-hIL-13Rα2 mouse monoclonal antibody (1/500 dilution, AF146, R&D system, Minneapolis, USA) or anti-TMEM219 polyclonal antibody (1/500 dilution, sc-244404, Santa Cruz Biotechnology Inc., CA, USA).

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: Lysates from these cells were subjected to immunoprecipitation using anti-hIL-13Rα2 mouse monoclonal antibody (1/500 dilution, AF146, R&D system, Minneapolis, USA) or anti-TMEM219 polyclonal antibody (1/500 dilution, sc-244404, Santa Cruz Biotechnology Inc., CA, USA).

Techniques: Western Blot, Activation Assay, Mutagenesis, Phospho-proteomics, 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: Lysates from these cells were subjected to immunoprecipitation using anti-hIL-13Rα2 mouse monoclonal antibody (1/500 dilution, AF146, R&D system, Minneapolis, USA) or anti-TMEM219 polyclonal antibody (1/500 dilution, sc-244404, Santa Cruz Biotechnology Inc., CA, USA).

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