Duolink in situ detection kit

The in situ proximity ligation assay (PLA) (26 (link)) was performed with the DuoLink in situ detection kit (Sigma) to detect and quantify the interaction of NPM with TLR4. Cells were grown on slides in 35 mm plates, fixed with 4% paraformaldehyde solution for 10 min, rinsed with PBS, and incubated for 1 h in the blocking buffer. α-NPM1 (ab15440; 1:400) or α-TLR4 (sc-293072; 1:100), α-rabbit PLUS/MINUS, and α-mouse PLUS/MINUS PLA probes were incubated for 1 h at 37°C and then washed. Hybridization, ligation, and amplification steps were performed for 30 min and polymerase reaction for 120 min at 37°C. Nuclei were counter-stained with DAPI (1:10,000 in PBS; Sigma). Images were captured with the ApoTome System (Zeiss) as described above. HFs and HaCaT cells were seeded on square glass coverslips. Subsequently, coverslips were removed and processed for PLA.
The PLA was quantified by counting the red dots and diving it for the number of nuclei per field, identified by DAPI staining by two independent observers, blinded to the status of the specimens. For each experiment, ~80–100 nuclei per treatment were accomplished. Six different primary HFs derived from healthy subjects and 5 different HaCaT experiments were performed.

The in situ proximity ligation assay (PLA) was performed using Duolink In Situ Detection kit (Sigma-Aldrich) as described previously [19 (link)]. Each red spot represents a cluster of protein-protein interaction, and distinct red spots were detected using confocal fluorescence microscope. The histogram quantifies the number of red spots per cell (n = 50 cells in two technical replicates). The results are representative of three independent experiments.

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)].

Interactions between different proteins were investigated with a Duolink® in situ detection kit (Sigma) as described previously (Shi et al. 2017a). Single VSMCs were fixed and permeabilized as per immunofluorescence and blocking was performed with Duolink blocking buffer for 1 h at 37°C. Cells were then incubated with appropriate antibodies overnight at 4°C as per immunofluorescence experiments plus mouse anti‐P‐Ser (dilution 1:100; sc‐16B4; Santa Cruz Biotechnology), mouse anti‐P‐Thr (dilution 1:100; sc‐5267; Santa Cruz Biotechnology), mouse anti‐TRPC1 (dilution 1:100; sc‐133 076; Santa Cruz Biotechnology) and rabbit anti‐TRPC5 (T5E3), which was generated by GenScript using peptide sequences from a previously characterized putative extracellular region (1 μg mL⁻¹) ((Xu & Beech, 2001; Xu et al. 2005a, 2005ba,b). The rest of the protocol was conducted in accordance with the manufacturer's instructions. Fluorescent puncta were visualized with an A1R confocal microscope and images were analysed with ImageJ Fiji (https://fiji.sc). The mean number of puncta per cell was calculated by counting the number of particles across a z‐stack of the cell.