Flow cytometric reporter assays provide robust functional analysis of signaling complexes.

Autor:	Muusse TW; School of Chemistry and Molecular Biosciences and Australian Infectious Diseases Research Centre, The University of Queensland, Brisbane, QLD, Australia., Lee MYL; School of Chemistry and Molecular Biosciences and Australian Infectious Diseases Research Centre, The University of Queensland, Brisbane, QLD, Australia., Kim H; School of Chemistry and Molecular Biosciences and Australian Infectious Diseases Research Centre, The University of Queensland, Brisbane, QLD, Australia., Parat MO; School of Pharmacy, The University of Queensland, Brisbane, QLD, Australia., Nanson JD; School of Chemistry and Molecular Biosciences and Australian Infectious Diseases Research Centre, The University of Queensland, Brisbane, QLD, Australia., Kobe B; School of Chemistry and Molecular Biosciences and Australian Infectious Diseases Research Centre, The University of Queensland, Brisbane, QLD, Australia., Vajjhala PR; School of Chemistry and Molecular Biosciences and Australian Infectious Diseases Research Centre, The University of Queensland, Brisbane, QLD, Australia. Electronic address: p.vajjhala@uq.edu.au., Stacey KJ; School of Chemistry and Molecular Biosciences and Australian Infectious Diseases Research Centre, The University of Queensland, Brisbane, QLD, Australia. Electronic address: katryn.stacey@uq.edu.au.
Jazyk:	angličtina
Zdroj:	The Journal of biological chemistry [J Biol Chem] 2022 Dec; Vol. 298 (12), pp. 102666. Date of Electronic Publication: 2022 Nov 02.
DOI:	10.1016/j.jbc.2022.102666
Abstrakt:	Conventional assays to probe signaling protein interactions and function involve measurement of luciferase reporter expression within the bulk cell population, with lack of control over target-protein expression level. To address this issue, we have developed a rapid and robust flow cytometric assay for analysis of signaling protein function. A fluorescent reporter and fluorescent tagging of the target protein enables simultaneous assessment of protein expression and signaling within individual cells. We have applied our technique to the analysis of variants of the lipopolysaccharide receptor Toll-like receptor 4 (TLR4) and its adapter protein MyD88, using a NF-кB-responsive promoter driving mScarlet-I expression. The assay enables exclusion of nontransfected cells and overexpressing cells that signal spontaneously. Additionally, our assay allows the identification of protein variants that fail to express. We found that the assays were highly sensitive, with cells expressing an appropriate level of GFP-MyD88 showing approximately 200-fold induction of mScarlet-I by lipopolysaccharide, and we can detect subtle protein concentration-dependent effects of mutations. Importantly, the assay is adaptable to various signaling pathways. Competing Interests: Conflict of interest The authors declare that they have no competing interests. (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
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