Quantitative in situ proximity ligation assays examining protein interactions
and phosphorylation during smooth muscle contractions.
Authors Xie Y, Perrino BA
Submitted By Submitted Externally on 5/13/2019
Status Published
Journal Analytical biochemistry
Year 2019
Date Published 4/1/2019
Volume : Pages 577 : 1 - 13
PubMed Reference 30981700
Abstract Antibody-based in situ proximity ligation assays (isPLA) have the potential to
study protein phosphorylation and protein interactions with spatial resolution
in intact tissues. However, the application of isPLA at the tissue level is
limited by a lack of appropriate positive and negative controls and the
difficulty in accounting for changes in tissue shape. Here we demonstrate a set
of experimental and computational approaches using gastric fundus smooth muscles
to improve the validity of quantitative isPLA. Appropriate positive and negative
biological controls and PLA technical controls were selected to ensure
experimental rigor. To account for changes in morphology between relaxed and
contracted smooth muscles, target PLA spots were normalized to smooth muscle
myosin light chain 20 PLA spots or the cellular cross-sectional areas. We
describe the computational steps necessary to filter out false-positive
improperly sized spots and set the thresholds for counting true positive PLA
spots to quantify the PLA signals. We tested our approach by examining protein
phosphorylation and protein interactions in smooth muscle myofilament Ca2+
sensitization pathways from resting and contracted gastric fundus smooth
muscles. In conclusion, our tissue-level isPLA method enables unbiased
quantitation of protein phosphorylation and protein-protein interactions in
intact smooth muscle tissues, suggesting the potential for quantitative isPLA
applications in other types of intact tissues.

No Complications