ArticleCurrent protocols2026
Protocols for Monitoring Unconventional Protein Secretion Using Luminescence and Trapping Approaches.
Article in Current protocols, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 1 paper.
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Who cites it
1 citing paper in PubMed.
- Protocols for Monitoring Unconventional Protein Secretion Using Luminescence and Trapping Approaches.Current protocols · 2026Article
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Authors and funding
8 authors.
Funding
Abstract
Unconventional protein secretion (UcPS) enables the export of cytosolic proteins through pathways that bypass the canonical endoplasmic reticulum-Golgi secretory route. Although increasingly recognized as essential for intercellular communication, stress responses, and tissue homeostasis, UcPS remains difficult to quantify due to low secretion efficiency, high intracellular background, and the challenge of distinguishing active secretion from passive leakage. Recent methodological advances, including NanoLuc split luciferase-based reporters and the Retention Using Selective Hooks (RUSH) system for synchronized protein transport, have improved sensitivity and temporal control of trafficking. Here, we present complementary protocols integrating these tools to provide a highly sensitive, quantitative workflow centered on a split NanoLuc (HiBiT/LgBiT) complementation assay for monitoring UcPS in mammalian cells. The Basic Protocol describes a robust luminescence-based secretion assay, while the Support Protocols detail the generation of stable HiBiT reporter cell lines, approaches for probing UcPS mechanisms using siRNA-mediated gene knockdown and pharmacological perturbation, and the incorporation of the RUSH system to synchronize cargo release and identify potential trafficking intermediates. Together, these protocols provide a sensitive, scalable, high-throughput toolkit that enables analysis of UcPS mechanisms across diverse cargo proteins, cell types, and perturbations. This methodological framework allows for rigorous dissection of UcPS pathways in both physiological and disease-relevant contexts. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Split luciferase complementation assay for quantifying UcPS in mammalian cells Support Protocol 1: Generation of stable cell lines expressing HiBiT-tagged cargo proteins for the split luciferase assay Support Protocol 2: siRNA-mediated knockdown to assess the role of candidate genes in UcPS Support Protocol 3: Pharmacological perturbation of UcPS Support Protocol 4: Integration of the RUSH system to synchronize UcPS.
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Registered trials
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