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Image-based high-throughput mapping of TGF-β-induced phosphocomplexes at a single-cell level.

Authors :
Lönn P
Al-Amin RA
Doulabi EM
Heldin J
Gallini R
Björkesten J
Oelrich J
Kamali-Moghaddam M
Landegren U
Source :
Communications biology [Commun Biol] 2021 Nov 12; Vol. 4 (1), pp. 1284. Date of Electronic Publication: 2021 Nov 12.
Publication Year :
2021

Abstract

Protein interactions and posttranslational modifications orchestrate cellular responses to e.g. cytokines and drugs, but it has been difficult to monitor these dynamic events in high-throughput. Here, we describe a semi-automated system for large-scale in situ proximity ligation assays (isPLA), combining isPLA in microtiter wells with automated microscopy and computer-based image analysis. Phosphorylations and interactions are digitally recorded along with subcellular morphological features. We investigated TGF-β-responsive Smad2 linker phosphorylations and complex formations over time and across millions of individual cells, and we relate these events to cell cycle progression and local cell crowding via measurements of DNA content and nuclear size of individual cells, and of their relative positions. We illustrate the suitability of this protocol to screen for drug effects using phosphatase inhibitors. Our approach expands the scope for image-based single cell analyses by combining observations of protein interactions and modifications with morphological details of individual cells at high throughput.<br /> (© 2021. The Author(s).)

Details

Language :
English
ISSN :
2399-3642
Volume :
4
Issue :
1
Database :
MEDLINE
Journal :
Communications biology
Publication Type :
Academic Journal
Accession number :
34773084
Full Text :
https://doi.org/10.1038/s42003-021-02798-4