Experimental Hematology, Department of Pediatrics, University Clinic Carl Gustav Carus, Dresden, Germany.
Molecular Analysis - Mass Spectrometry, Center for Molecular and Cellular Bioengineering (CMCB), Technische Universitaet Dresden, Dresden, Germany.
Front Immunol. 2021 Apr 16;12:648283. doi: 10.3389/fimmu.2021.648283. eCollection 2021.
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.
炎症状态受到神经免疫相互作用的严格影响。细胞因子和神经营养因子塑造了神经系统和免疫系统的反应。尽管已经取得了很大的进展，但迄今为止的大多数发现都是基于重组（标记）蛋白的表达。通过免疫沉淀（IP）在内源性水平检查受体相互作用，可进一步深入了解免疫反应的更微妙的调节。在这里，我们提出了一个全面的工作流程和优化的 IP 协议，为研究神经生长因子受体 p75NTR 在低丰度的内源性水平提供了分步说明：从裂解物制备和受体表达的确认到抗体验证和成功检测蛋白质-蛋白质相互作用。我们使用人黑素瘤细胞系 A375 来验证特异性抗体和 IP 条件，并应用这些方法来探索人白血病浆细胞样树突状细胞系 PMDC05 中的 p75NTR 相互作用，检测到该细胞类型中 14-3-3ε：p75NTR 相互作用。以 p75NTR 为示例蛋白，我们的方法提供了一种策略，可以在低丰度的内源性表达条件下检测特定的相互作用伙伴。
