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

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 relati

SUBMITTER: Lonn P 

PROVIDER: S-EPMC8590043 | biostudies-literature | 2021 Nov

REPOSITORIES: biostudies-literature