Figure 1: TMEM219 physically interacts with IL-13Rα2.

(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.