TY - JOUR
T1 - Listening to the Whispers in Neuroimmune Crosstalk
T2 - A Comprehensive Workflow to Investigate Neurotrophin Receptor p75NTR Under Endogenous, Low Abundance Conditions
AU - Dorschner, Benjamin W.
AU - Wiedemuth, Ralf
AU - Funke, Ann Christin
AU - Gentzel, Marc
AU - Rogers, Mary Louise
AU - Brenner, Sebastian
AU - Thieme, Sebastian
PY - 2021/4/16
Y1 - 2021/4/16
N2 - 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.
AB - 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.
KW - A375 (human melanoma) cell line
KW - CD271 (p75NTR)
KW - endogenous immunoprecipitation
KW - mass spectrometry
KW - neuroimmune crosstalk
KW - p75NTR
KW - PMDC05
KW - protein-protein interaction
UR - http://www.scopus.com/inward/record.url?scp=85105164298&partnerID=8YFLogxK
U2 - 10.3389/fimmu.2021.648283
DO - 10.3389/fimmu.2021.648283
M3 - Article
C2 - 33936068
AN - SCOPUS:85105164298
SN - 1664-3224
VL - 12
JO - Frontiers in Immunology
JF - Frontiers in Immunology
M1 - 648283
ER -