Proximity Ligation Assay for Protein Interactions

Cells were seeded to a density of 50–70% on 12 mm glass coverslips coated in poly-L-lysine (Sigma-Aldrich, P8920). Cells were fixed with a 2% PFA solution (2% PFA, 0.2% Triton-X-100, pH 8.2), washed with 1X PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄), permeabilized with 0.5% tergitol (Sigma-Aldrich, NP40S), and washed with 1X PBS. PLAs were performed according to manufacturer’s instructions using the Duolink In Situ Detection Kit (Sigma-Aldrich, DUO92013). Confocal microscopy was done at the SickKids Imaging Facility and foci counted using CellProfiler [106 (link)]. Signal intensity was obtained using ImageJ, with background signal subtracted for each figure [107 (link)].