Listening to the Whispers in Neuroimmune Crosstalk: A Comprehensive Workflow to Investigate Neurotrophin Receptor p75NTR Under Endogenous, Low Abundance Conditions

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...

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Published inFrontiers in immunology Vol. 12; p. 648283
Main Authors Dorschner, Benjamin W, Wiedemuth, Ralf, Funke, Ann-Christin, Gentzel, Marc, Rogers, Mary-Louise, Brenner, Sebastian, Thieme, Sebastian
Format Journal Article
LanguageEnglish
Published Switzerland Frontiers Media S.A 16.04.2021
Subjects
CD271 (p75NTR)
endogenous immunoprecipitation
Immunology
mass spectrometry
p75NTR
PMDC05
protein-protein interaction
A375 (human melanoma) cell line
PMDC05
p75NTR
CD271 (p75NTR)
endogenous immunoprecipitation
mass spectrometry
protein-protein interaction
neuroimmune crosstalk
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Summary: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.
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Edited by: Jorge Correale, Fundación Para la Lucha Contra las Enfermedades Neurológicas de la Infancia (FLENI), Argentina
This article was submitted to Multiple Sclerosis and Neuroimmunology, a section of the journal Frontiers in Immunology
Reviewed by: Elizabeth J. Coulson, The University of Queensland, Australia; Bruce Douglas Carter, Vanderbilt University, United States; Rick Meeker, University of North Carolina at Chapel Hill, United States
ISSN:1664-3224
1664-3224
DOI:10.3389/fimmu.2021.648283