In situ proximity ligation assay

In situ proximity ligation assays (PLA) were performed as described previously to detect protein interactions (Huang et al. 2011; Zhang et al. 2012, 2016). PLA provides for the precise detection of protein–protein complexes (Soderberg et al. 2006, 2008). Target proteins are reacted with primary antibodies raised in different species, and a pair of oligonucleotide‐labelled secondary antibodies conjugated to + and − PLA probes is targeted to each pair of primary antibodies. The probes form circular DNA strands only when they are bound in very close proximity (< 40 nm). These DNA circles serve as templates for localized rolling circle amplification, generating a fluorescence signal (spot) that enables individual interacting pairs of the target protein molecules to be visualized. The PLA signal allows for the detection of a complex between two target proteins at a very high resolution.

Smooth muscle cells were dissociated from Sham‐treated or transfected canine tracheal smooth muscle tissues. Freshly dissociated smooth muscle cells were stimulated with 10⁻⁵ m ACh or left unstimulated and then fixed, permeabilized and incubated with primary antibodies against target proteins or epitopes followed by a pair of oligonucleotide‐labelled secondary antibodies conjugated to Duolink + and − PLA probes. PLA probe hybridization, ligation, amplification and detection media were administered in accordance with the manufacturer's instructions (Olink Bioscience, Uppsala, Sweden). Randomly selected cells from both unstimulated and ACh‐stimulated groups were analysed for protein interactions by visualizing PLA fluorescence spots using a LSM 510 confocal microscope (Carl Zeiss, Oberkochen, Germany).

The total number of PLA fluorescence spots/cell was counted using Image Tools (Olink Bioscience). In some experiments, the distribution of PLA fluorescence spots within each cell was quantified by separating the region of the cell cortex from the cytoplasm by a line ∼1 μm inside the cell border. The ratio of fluorescence intensity between the cortical compartment and the cytoplasmic compartment was calculated using Metamorph (Molecular Devices, Inc. Sunnyvale, CA, USA) as described previously (Zhang et al. 2012). The ratio of the area of cortical compartment to the area of the cytoplasmic compartment did not differ significantly in any of the groups.