Journal: Nature Communications

Article Title: Interleukin 31 receptor α promotes smooth muscle cell contraction and airway hyperresponsiveness in asthma

doi: 10.1038/s41467-023-44040-1

Figure Lengend Snippet: a Schemata of HDM-induced allergic asthma model. Image was created with biorender.com. b Quantification of IL-31RA transcripts in the lungs of wildtype ( n = 5) and IL-31RA –/– ( n = 5) mice treated with saline or wildtype ( n = 6) and IL-31RA –/– ( n = 6) mice treated with HDM. Data shown as mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm$$\end{document} ± SEM. Two-way ANOVA test was used. c Quantification of IL-31 transcripts in the lungs of wildtype ( n = 5) and IL-31RA ( n = 5) mice treated with saline or wildtype ( n = 5) and IL-31RA ( n = 6) mice treated with HDM. Data shown as mean ± SEM. Two-way ANOVA test was used. d Representative images of immunohistochemical staining for IL-31RA on lung sections from wild-type mice treated with saline ( n = 4) or HDM ( n = 4). Scale bar, 50 µm. e Representative images of immunohistochemical staining for IL-31RA on lung sections from healthy controls ( n = 8) and asthmatics ( n = 8). Arrow heads highlight the IL-31RA staining (brown) in smooth muscle cells with spindle-shaped nuclei. Scale bar, 50 µm. f Measurement of resistance with increasing doses of methacholine (MCh) in wildtype ( n = 7) and IL-31RA –/– ( n = 8) mice treated with saline or wildtype ( n = 5) and IL-31RA –/– ( n = 8) mice treated with HDM using FlexiVent. Data are shown as mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SEM. A two-way ANOVA test was used. g The percent of contraction of airways with increasing doses of MCh compared to the baseline area of airways between wild-type ( n = 8) and IL-31RA –/– ( n = 6) mice. A two-way ANOVA test was used. Data shown as mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SEM. h The percent contraction of collagen gels embedded with airway smooth muscle cells from wild-type ( n = 3) and IL-31RA –/– ( n = 3) mice in culture media and treated with carbachol (10 μM) for 60 min. Data shown as mean ± SEM. Unpaired t test was used. At least two independent experiments produced similar results. Source data are provided as a Source Data file.

Article Snippet: After SDS–PAGE separation, proteins were transferred to nitrocellulose membrane and incubated overnight with primary antibodies against CHRM3 (1:1000, Abcam), phosphoMLC (1:1000, Cell Signaling Technology), totalMLC (1:1000, Cell Signaling Technology), hIL-31RA (1:100, R&D Systems), ITGB1 (1:1000, Cell Signaling Technology) and GAPDH (1:2000, Bethyl Laboratories) and followed by detection with HRP conjugated secondary antibodies.

Techniques: Saline, Immunohistochemical staining, Staining, Produced

Journal: Nature Communications

Article Title: Interleukin 31 receptor α promotes smooth muscle cell contraction and airway hyperresponsiveness in asthma

doi: 10.1038/s41467-023-44040-1

Figure Lengend Snippet: a Representative image of hematoxylin and eosin-stained lung sections from wildtype ( n = 5) and IL-31RA –/– ( n = 5) mice treated with saline or HDM. Images were taken at ×20 magnification, scale bar 100 µm. b , c Total bronchoalveolar lavage (BAL) cell number and the differential cell count of BAL cells of wild-type and IL-31RA -/- mice treated with saline ( n = 5) or HDM ( n = 5). Eos, eosinophils; Mac, macrophages; Lym, lymphocytes; and Neu, neutrophils. One-way ANOVA was used. d Representative images of Alcian blue periodic acid-Schiff (ABPAS) staining of lung sections from wildtype ( n = 7) and IL-31RA –/– ( n = 7) mice treated with saline or HDM. Images were taken at ×20 magnification, scale bar 100 µm. e The percent of ABPAS-positive cells normalized to total cell in the airways of wild-type and IL-31RA –/– mice treated with saline ( n = 7) or HDM ( n = 7). Data are shown as mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SEM. One-way ANOVA was used. At least two independent experiments produced similar results. Source data are provided as a Source Data file.

Article Snippet: After SDS–PAGE separation, proteins were transferred to nitrocellulose membrane and incubated overnight with primary antibodies against CHRM3 (1:1000, Abcam), phosphoMLC (1:1000, Cell Signaling Technology), totalMLC (1:1000, Cell Signaling Technology), hIL-31RA (1:100, R&D Systems), ITGB1 (1:1000, Cell Signaling Technology) and GAPDH (1:2000, Bethyl Laboratories) and followed by detection with HRP conjugated secondary antibodies.

Techniques: Staining, Saline, Cell Counting, Produced

Journal: Nature Communications

Article Title: Interleukin 31 receptor α promotes smooth muscle cell contraction and airway hyperresponsiveness in asthma

doi: 10.1038/s41467-023-44040-1

Figure Lengend Snippet: a Quantification of Th2 cytokine transcripts including IL-4, IL5, and IL-13 in the whole lung tissue of wild-type and IL-31RA -/- mice treated with saline ( n = 6) or HDM ( n = 6) using RT-PCR. Data are shown as mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SEM. One-way ANOVA was used. b Quantification of chemokines and inflammatory cytokines including CCL11, CCL24, and IL-10 in the whole lungs of wild-type and IL-31RA -/- mice treated with saline ( n = 6) or HDM ( n = 6) using RT-PCR. Data are shown as mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SEM. One-way ANOVA was used. c Quantification of Th2 response-associated gene transcripts including CHI3L3, ARG1, and FIZZ1 in the total lungs of wild-type and IL-31RA –/– mice treated with saline ( n = 6) or HDM ( n = 6) using RT-PCR. Data are shown as mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SEM. One-way ANOVA was used. d Quantification of GOB5 and MUC5AC gene transcripts in the whole lungs of wild-type and IL-31RA –/– mice treated with saline ( n = 6) or HDM ( n = 6) using RT-PCR. Data are shown as mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SEM. One-way ANOVA was used. e Concentration of cytokines IL-4, IL-13, IL-5, IL-6, IFNγ, TNF-α and IL-10 in the BAL fluids of wild-type and IL-31RA –/– mice treated with saline ( n = 6) or HDM ( n = 6) using the cytometric bead assay LEGENDplex. Data are shown as mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SEM. One-way ANOVA was used. At least two independent experiments produced similar results. Source data are provided as a Source Data file.

Article Snippet: After SDS–PAGE separation, proteins were transferred to nitrocellulose membrane and incubated overnight with primary antibodies against CHRM3 (1:1000, Abcam), phosphoMLC (1:1000, Cell Signaling Technology), totalMLC (1:1000, Cell Signaling Technology), hIL-31RA (1:100, R&D Systems), ITGB1 (1:1000, Cell Signaling Technology) and GAPDH (1:2000, Bethyl Laboratories) and followed by detection with HRP conjugated secondary antibodies.

Techniques: Saline, Reverse Transcription Polymerase Chain Reaction, Concentration Assay, Produced

Journal: Nature Communications

Article Title: Interleukin 31 receptor α promotes smooth muscle cell contraction and airway hyperresponsiveness in asthma

doi: 10.1038/s41467-023-44040-1

Figure Lengend Snippet: a Schemata of SEA-induced allergic asthma model. Image was created with biorender.com. b Quantification of IL-31RA transcripts in the lungs of wildtype ( n = 5) and IL-31RA –/– ( n = 6) mice treated with saline or wild-type ( n = 6) and IL-31RA –/– ( n = 6) mice treated with SEA. Data shown as mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SEM. Two-way ANOVA test was used. c Quantification of IL-31 transcripts in the lungs of wildtype ( n = 5) and IL-31RA –/– ( n = 5) mice treated with saline or wild-type ( n = 6) and IL-31RA –/– ( n = 5) mice treated with SEA. Data shown as mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SEM. Two-way ANOVA test was used. d , e Measurement of resistance with increasing doses of methacholine (MCh) in wildtype ( n = 6) and IL-31RA –/– ( n = 7) mice treated with saline or wildtype ( n = 5) and IL-31RA –/– ( n = 4) mice treated with SEA using FlexiVent. Data are shown as mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SEM. A two-way ANOVA test was used. f Representative images of hematoxylin and eosin-stained lung sections from wildtype ( n = 5) and IL-31RA –/– ( n = 5) mice treated with saline or SEA. Images were taken at ×20 magnification. Scale bar, 100 µm. g Representative images of Alcian blue periodic acid-Schiff staining of lung sections from wildtype ( n = 5) and IL-31RA –/– ( n = 5) mice treated with saline or SEA. Images were taken at ×20 magnification. Scale bar, 100 µm. At least two independent experiments produced similar results. Source data are provided as a Source Data file.

Article Snippet: After SDS–PAGE separation, proteins were transferred to nitrocellulose membrane and incubated overnight with primary antibodies against CHRM3 (1:1000, Abcam), phosphoMLC (1:1000, Cell Signaling Technology), totalMLC (1:1000, Cell Signaling Technology), hIL-31RA (1:100, R&D Systems), ITGB1 (1:1000, Cell Signaling Technology) and GAPDH (1:2000, Bethyl Laboratories) and followed by detection with HRP conjugated secondary antibodies.

Techniques: Saline, Staining, Produced

Journal: Nature Communications

Article Title: Interleukin 31 receptor α promotes smooth muscle cell contraction and airway hyperresponsiveness in asthma

doi: 10.1038/s41467-023-44040-1

Figure Lengend Snippet: a , b Quantification of IL-31RA transcripts in mouse ASMC treated with increasing doses of IL-4 ( n = 3) and IL-13 ( n = 4) for 16 h. Two-way ANOVA test was used. Data shown as mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SEM. c Wildtype ( n = 4) and IL-31RA –/– ( n = 4) mice were treated intratracheally with IL-13 on days 0 and 6, and resistance in the lungs was measured with increasing doses of methacholine (MCh) using FexiVent. Data are shown as mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SEM. Two-way ANOVA test was used. d ASMC isolated from wildtype ( n = 3) and IL-31RA –/– ( n = 3) mice were seeded into collagen gels and treated with IL-13 to measure the contraction of collagen gels after 48 h. Unpaired t test was used. Data shown as mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SEM. e Representative images of hematoxylin and eosin -stained lung sections from wildtype ( n = 4) and IL-31RA –/– ( n = 4) mice treated with IL-13. Images were captured at ×20 magnification. Scale bar, 100 µm. f , g , h Quantification of inflammatory chemokines ( CCL11, CCL24 and IL-17 ), Th2 cytokines ( IL-4 and IL-5 ), and Th2 response-associated genes ( ARG1, CHl3L3 , and FIZZ1 ) in the whole lungs of wildtype ( n = 4) and IL-31RA –/– ( n = 4) mice treated with IL-13. Data are shown as mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SEM. Unpaired t test was used, and no statistical significance observed between groups. i Representative images of Alcian blue periodic acid-Schiff-stained lung sections from wildtype ( n = 4) and IL-31RA –/– ( n = 4) mice treated with IL-13. Images were captured at ×20 magnification. Scale bar, 100 µm. j Quantification of goblet cell hyperplasia associated genes including GOB5 and MUC5AC transcript levels in the total lungs of wildtype ( n = 4) and IL-31RA –/– ( n = 4) mice treated with IL-13. Data are shown as means \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SEM. Unpaired t test was used, and no statistical significance observed between groups. At least two independent experiments produced similar results. Source data are provided as a Source Data file.

Article Snippet: After SDS–PAGE separation, proteins were transferred to nitrocellulose membrane and incubated overnight with primary antibodies against CHRM3 (1:1000, Abcam), phosphoMLC (1:1000, Cell Signaling Technology), totalMLC (1:1000, Cell Signaling Technology), hIL-31RA (1:100, R&D Systems), ITGB1 (1:1000, Cell Signaling Technology) and GAPDH (1:2000, Bethyl Laboratories) and followed by detection with HRP conjugated secondary antibodies.

Techniques: Isolation, Staining, Produced

Journal: Nature Communications

Article Title: Interleukin 31 receptor α promotes smooth muscle cell contraction and airway hyperresponsiveness in asthma

doi: 10.1038/s41467-023-44040-1

Figure Lengend Snippet: a Quantification of IL-31RA transcript levels in mouse airway smooth muscle cells (ASMC) treated with increasing doses of IFNγ ( n = 3) or media ( n = 3) for 16 h. Data are shown as the mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SEM. Two-way ANOVA was used. b Quantification of IL-31RA transcript levels in human ASMCs treated with media, IL-31 (500 ng/mL) or IFNγ (50 ng/mL) for 16 h ( n = 4). Data are shown as the mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SEM. Two -way ANOVA was used. c Wildtype mice were intratracheally treated with saline ( n = 4) or IFNγ (5 µg) ( n = 4) on days 0 and 6, and airway resistance with increasing doses of methacholine was measured on day 7. Data are shown as mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SEM. Two-way ANOVA was used. d Representative images of hematoxylin and eosin -stained lung sections from wildtype mice treated with saline ( n = 4) or IFNγ ( n = 4). Images were captured at ×20 magnification. Scale bar, 100 µm. e , f , g Quantification of inflammatory chemokines ( CCL11 and IL-17 ), Th2 cytokines ( IL-4, IL-5, and CCL24) , and Th2 response-associated genes, including ARG1 and CHl3L3 in the lungs of wildtype mice treated with saline ( n = 4) or IFNγ ( n = 4). Data are shown as the mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SEM. Unpaired t test was used. h Representative images of Alcian blue periodic acid-Schiff-stained lung sections from wild-type mice treated with saline ( n = 4) or IFNγ ( n = 4). Images were captured at ×20 magnification. Scale bar, 100 µm. i Quantification of mucus-associated gene transcripts, including GOB5 and MUC5AC , in the lungs of wildtype mice treated with saline ( n = 4) or IFNγ ( n = 4). Data are shown as the mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SEM. Unpaired t test was used. At least two independent experiments produced similar results. Source data are provided as a Source Data file.

Article Snippet: After SDS–PAGE separation, proteins were transferred to nitrocellulose membrane and incubated overnight with primary antibodies against CHRM3 (1:1000, Abcam), phosphoMLC (1:1000, Cell Signaling Technology), totalMLC (1:1000, Cell Signaling Technology), hIL-31RA (1:100, R&D Systems), ITGB1 (1:1000, Cell Signaling Technology) and GAPDH (1:2000, Bethyl Laboratories) and followed by detection with HRP conjugated secondary antibodies.

Techniques: Saline, Staining, Produced

Journal: Nature Communications

Article Title: Interleukin 31 receptor α promotes smooth muscle cell contraction and airway hyperresponsiveness in asthma

doi: 10.1038/s41467-023-44040-1

Figure Lengend Snippet: a Wildtype ( n = 9) and IL-31RA –/– ( n = 9) mice were treated intratracheally with IFNγ (5 μg) on days 0 and 6, and resistance was measured with increasing doses of MCh using Fexivent. Data are shown as mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SEM. Two-way ANOVA was used. b Representative images of hematoxylin and eosin-stained lung sections from wildtype ( n = 6) and IL-31RA –/– ( n = 6) mice treated with IFNγ. Images were captured at ×20 magnification. Scale bar, 100 µm. c , d , e Quantification of inflammatory cytokines ( CCL11, CCL24 and IL-17) , Th2 cytokines ( IL-4 and IL-5) , and Th2 response-associated genes including ARG1, and CHl3L3 in the lungs of wildtype ( n = 6) and IL-31RA –/– ( n = 6) mice treated with IFNγ. Data are shown as mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SEM. Unpaired t test was used, and no statistical signification observed between groups. f Representative images of Alcian blue periodic acid-Schiff-stained lung sections from wildtype ( n = 6) and IL-31RA –/– ( n = 6) mice treated with IFNγ. Images were captured at ×20 magnification. Scale bar, 100 µm. g Quantification of mucus-associated genes including GOB5 and MUC5AC transcript levels in the lungs of wildtype ( n = 6) and IL-31RA –/– ( n = 6) mice treated with IFNγ. Data are shown as means \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SEM. Unpaired t test was used, and no statistical signification observed between groups. At least two independent experiments produced similar results. Source data are provided as a Source Data file.

Article Snippet: After SDS–PAGE separation, proteins were transferred to nitrocellulose membrane and incubated overnight with primary antibodies against CHRM3 (1:1000, Abcam), phosphoMLC (1:1000, Cell Signaling Technology), totalMLC (1:1000, Cell Signaling Technology), hIL-31RA (1:100, R&D Systems), ITGB1 (1:1000, Cell Signaling Technology) and GAPDH (1:2000, Bethyl Laboratories) and followed by detection with HRP conjugated secondary antibodies.

Techniques: Staining, Produced

Journal: Nature Communications

Article Title: Interleukin 31 receptor α promotes smooth muscle cell contraction and airway hyperresponsiveness in asthma

doi: 10.1038/s41467-023-44040-1

Figure Lengend Snippet: a Quantification of CHRMs transcripts in the lungs of wildtype ( n = 5) and IL-31RA –/– ( n = 5) mice using RT-PCR. Data shown as mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SEM. Two-way ANOVA was used. b, c Quantification of CHRM3 transcripts in ASMC isolated from wildtype ( n = 3) and IL-31RA –/– ( n = 3) mice and treated with media, IL-4 (10 ng/mL) or IL-31 (500 ng/mL) for 16 h. Data shown as mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SEM. Two-way ANOVA was used. d , e The total lung lysates of HDM-treated wildtype ( n = 5) and IL-31RA –/– ( n = 5) mice were immunoblotted with antibodies against CHRM3 and GAPDH. Data are shown as mean ± SEM. Unpaired t test was used. f ASMC isolated from wildtype ( n = 3) and IL-31RA –/– ( n = 3) mice were lysed and immunoblotted with antibodies against CHRM3 and GAPDH. Data are shown as mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SEM. Unpaired t test was used. g hTERT cells were transiently transfected with IL-31RA-specific ( n = 3) or control siRNA ( n = 3) for 72 h. Cell lysates were immunoblotted with antibodies against IL-31RA, CHRM3 and β-actin. Bar graph shows CHRM3 protein levels normalized to β-actin. Unpaired t test was used. Data are shown as means ± SEM. h Cell surface proteins were biotinylated, and affinity purified using neutravidin to measure cell surface levels of IL-31RA and CHRM3 in hTERT cells transfected with IL31RA-specific ( n = 2) or control siRNA ( n = 2) for 72 h. hTERT cells without biotinylation were used as a negative control. CHRM3 protein levels were normalized to ITGB1. i HEK293 cell were transiently transfected with overexpressing plasmids for CHRM3 and IL-31RA or empty control plasmids for 48 h. Cells were treated with antibodies against IL-31RA and CHRM3 and the IL-31RA-CHRM3 complex formation was visualized using hybridization probes at an excitation λex 594 nm (Red). The plasma membrane was stained with cholera toxin subunit b conjugated with Alexa Fluor 488 (Green) and the nuclei with DAPI (Blue). The white arrowheads highlight colocalization between the cholera toxin and puncta of the IL-31RA-CHRM3 complex. Images were captured at ×40 magnification. Scale bar, 50 µm. j hTERT cells were treated with antibodies against IL-31RA and CHRM3 or isotype IgG (negative PLA staining). The IL-31RA-CHRM3 complex formation was visualized as described in Fig. 9i. k HEK293T cells transiently transfected with a control plasmid or FLAG-tagged IL-31RA OE plasmid for 72 h. Total cell lysates and eluted fractions were immunoblotted with anti-Flag, anti-IL31RA and anti-CHRM3 antibodies. At least two independent experiments produced similar results. Source data are provided as a Source Data file.

Article Snippet: After SDS–PAGE separation, proteins were transferred to nitrocellulose membrane and incubated overnight with primary antibodies against CHRM3 (1:1000, Abcam), phosphoMLC (1:1000, Cell Signaling Technology), totalMLC (1:1000, Cell Signaling Technology), hIL-31RA (1:100, R&D Systems), ITGB1 (1:1000, Cell Signaling Technology) and GAPDH (1:2000, Bethyl Laboratories) and followed by detection with HRP conjugated secondary antibodies.

Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation, Transfection, Control, Affinity Purification, Negative Control, Hybridization, Clinical Proteomics, Membrane, Staining, Plasmid Preparation, Produced

Journal: Nature Communications

Article Title: Interleukin 31 receptor α promotes smooth muscle cell contraction and airway hyperresponsiveness in asthma

doi: 10.1038/s41467-023-44040-1

Figure Lengend Snippet: a Intracellular calcium levels were measured using Fluo-4 AM dye in HEK293T cells transiently transfected with control or overexpressing plasmids for CHRM3 and/or IL-31RA plasmids for 72 h and treated with 10 µM carbachol ( n = 4). b Intracellular calcium levels were measured using Fluo-4 AM dye in HEK293T cells transiently transfected with control or overexpressing plasmid for IL-31RA for 72 h and treated with 50 µM bradykinin ( n = 3). c , d , e , f Intracellular calcium levels were measured using Fluo-4 AM dye in hTERT cells transiently transfected with control or overexpressing plasmid for IL-31RA for 72 h and cells treated with 10 µM carbachol ( n = 8), 10 µM bradykinin ( n = 8), 100 µM serotonin ( n = 4) or 10 µM ionomycin ( n = 4). g hTERT cell line 72 (CHRM3 LOW ) was transiently transfected with control or IL-31RA overexpression plasmids for 72 h and intracellular calcium levels were measured in cells treated with 10 µM carbachol ( n = 5) or 10 µM ionomycin ( n = 3) using Fluo 4 AM dye. Data are shown as means ± SEM. h Intracellular calcium levels were measured using Fluo-4 AM dye in hTERT cells transiently transfected with IL-31RA-specific or control siRNA for 72 h and treated with 10 µM carbachol ( n = 8). i hTERT cells were transiently transfected with IL-31RA overexpressing plasmid for 48 h and treated with IL-31 (500 ng/mL) or media for another 24 h. Media or IL-31 treated cells were stimulated with 10 µM carbachol ( n = 4) and intracellular calcium levels were measured using Fluo-4 AM dye. Data are shown as means ± SEM. j hTERT cells were transiently transfected with IL-31RA-specific or control siRNA for 72 h and treated with 10 µM carbachol ( n = 3) for 10 min. Cell lysates were immunoblotted with antibodies against phospho-MLC, and β-actin. Data are shown as means \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SEM. Unpaired t test was used. k ASMC isolated from wildtype and IL-31RA –/– mice were treated with 10 µM carbachol ( n = 3) for 10 min and cell lysates were immunoblotted with antibodies against phospho-MLC, total-MLC and GAPDH. Data are shown as means \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SEM. Unpaired t test was used. At least two independent experiments produced similar results. Source data are provided as a Source Data file.

Article Snippet: After SDS–PAGE separation, proteins were transferred to nitrocellulose membrane and incubated overnight with primary antibodies against CHRM3 (1:1000, Abcam), phosphoMLC (1:1000, Cell Signaling Technology), totalMLC (1:1000, Cell Signaling Technology), hIL-31RA (1:100, R&D Systems), ITGB1 (1:1000, Cell Signaling Technology) and GAPDH (1:2000, Bethyl Laboratories) and followed by detection with HRP conjugated secondary antibodies.

Techniques: Transfection, Control, Plasmid Preparation, Over Expression, Isolation, Produced