1. Listening to the Whispers in Neuroimmune Crosstalk: A Comprehensive Workflow to Investigate Neurotrophin Receptor p75NTR Under Endogenous, Low Abundance Conditions
- Author
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Ralf Wiedemuth, Ann-Christin Funke, Marc Gentzel, Mary-Louise Rogers, Benjamin Dorschner, Sebastian Thieme, and Sebastian Brenner
- Subjects
0301 basic medicine ,Immunoprecipitation ,Receptor expression ,Blotting, Western ,Immunology ,Fluorescent Antibody Technique ,Gene Expression ,Nerve Tissue Proteins ,Endogeny ,Receptors, Nerve Growth Factor ,Plasmacytoid dendritic cell ,Biology ,Real-Time Polymerase Chain Reaction ,Antibodies ,Mass Spectrometry ,Workflow ,endogenous immunoprecipitation ,protein-protein interaction ,Mice ,p75NTR ,03 medical and health sciences ,0302 clinical medicine ,Neurotrophic factors ,Cell Line, Tumor ,Methods ,Animals ,Humans ,Immunology and Allergy ,RNA, Messenger ,neuroimmune crosstalk ,Receptor ,Hybridomas ,A375 (human melanoma) cell line ,Dendritic Cells ,RC581-607 ,Flow Cytometry ,CD271 (p75NTR) ,Cell biology ,Crosstalk (biology) ,030104 developmental biology ,14-3-3 Proteins ,030220 oncology & carcinogenesis ,biology.protein ,Immunologic diseases. Allergy ,PMDC05 ,Protein Binding ,Neurotrophin - Abstract
Inflammatory conditions are critically influenced by neuroimmune crosstalk. Cytokines and neurotrophic factors shape the responses of both nervous and immune systems. Although much progress has been made, most findings to date are based on expression of recombinant (tagged) proteins. The examination of receptor interactions by immunoprecipitation (IP) at endogenous levels provides further insight into the more subtle regulations of immune responses. Here, we present a comprehensive workflow and an optimized IP protocol that provide step-by-step instructions to investigate neurotrophin receptor p75NTR at endogenous, low abundance levels: from lysate preparation and confirmation of receptor expression to antibody validation and successful detection of protein-protein interactions. We employ human melanoma cell line A375 to validate specific antibodies and IP conditions, and apply these methods to explore p75NTR interactions in human leukemic plasmacytoid dendritic cell line PMDC05 detecting 14-3-3ϵ:p75NTR interaction in this cell type. With p75NTR as an exemplary protein, our approach provides a strategy to detect specific interaction partners even under endogenous, low abundance expression conditions.
- Published
- 2021