Duolink kit

For in situ PLA experiments, the DuoLink kit (Olink Bioscience) was used according to manufacturer's recommendations. Briefly, DLD1 cells were rehydrated and permeabilized as described for proxHCR. On blocking of nonspecific binding sites with DuoLink blocking reagent for 1 h at 37 °C, we incubated cells with primary antibodies directed against E-cadherin (1:100) and β-catenin (1:300) overnight at 4 °C. Cells were washed with DuoLink Wash buffer A two time for 5 min and incubated with proximity probes (PLUS and MINUS, Olink Bioscience) in antibody diluent (Olink Bioscience) for 1 h at 37 °C. After a new round of washing steps, ligation mix was applied to the cells and incubated for 30 min at 37 °C. Additional washing was followed by RCA and fluorescent detection of the rolling circle products. Cells were counterstained with Hoechst and mounted as described above.

In situ PLAs were performed to quantify protein-protein interactions between NCS-1 and IP₃Rs in cells using the Duolink kit (O-link Bioscience, Uppsala, Sweden). Using this protocol, only when the two target proteins are in close proximity (<40 nm), high concentration of fluorescence amplified from each interacted molecule becomes visible as a distinct bright dot when viewed with a fluorescence microscope. Briefly, cultured NMVMs were fixed, permeabilized, and blocked. The cells were then incubated with primary antibodies against hemagglutinin (HA; to detect of NCS-1-HA) and IP₃RI-III, followed by the appropriate secondary antibodies containing unique DNA strands (called PLA probes). Anti-α-actinin antibody followed by Alexa Fluor 488-conjugated anti-mouse secondary antibody (Life Technologies) and 4′,6-diamidino-2-phenylindole (DAPI) solution (Dojindo, Kumamoto, Japan) were added to visualize the myocytes and nuclei, respectively. The samples were evaluated by confocal microscopy. Images were analyzed by the ImageJ software (NIH) using the Cell Counter plug-in to count the number of PLA signals for each α-actinin-positive cell.

PLA was performed according to the manufacturer’s protocol (Duolink kit, Olink Bioscience, Uppsala, Sweden). Briefly, HEK293T cells were grown on coverslips and fixed at room temperature (RT) with 3.7% formaldehyde for 20 min. After three washes with phosphate-buffered saline (PBS), cells were blocked and permeabilized for 60 min in 5% BSA/0.3% Triton-X100 at RT and incubated with primary antibodies at optimized dilutions (rabbit anti-ROQUIN 1:75 (Novus Biologicals); with either mouse anti-RCK 1:150 (Santa Cruz Biotechnology Inc.); mouse anti-Ago2 1:200 (WAKO Pure Chemical Industries) or mouse anti-GFP 1:100 (Roche)) overnight in a humid chamber at 4 °C. Slides were washed three times in PBS/0.05% Tween20 and incubated with mouse minus and rabbit plus PLA probes for 1 h at 37 °C. Ligation was carried out for 30 min and amplification for 100 min at 37 °C. Slides were washed, dried at RT in the dark and mounted in mounting media with DAPI (Olink) to stain nuclei. The images were taken on a Leica SP5 confocal microscope with a pin hole of 95.5 μm and a HCxPL APO lambda blue × 631.4 oil objective and quantified using the free software ImageJ (NIH, Maryland, USA).

Cells were deposited on glass slides and fixed with methanol for 10 min. PLA was performed using the Duolink kit (Olink Bioscience, Sweden), according to manufacturer’s recommendations. The following combination of primary anti-human antibodies were used against: P-cadherin (dilution 1:50, rabbit polyclonal IgG, Cell Signaling), E-cadherin (dilution 1:100, mouse monoclonal IgG1, clone HECD-1, Takara Bio Inc., Shiga, Japan; or dilution 1:50, rabbit monoclonal IgG, Cell Signaling), p120ctn (dilution 1:100, mouse monoclonal IgG1, clone 98, BD Biosciences). The nuclei were counterstained with DAPI. Slides were analyzed with fluorescence microscopy (Zeiss Imager Z1 microscope) for visualization of bright fluorescent red signals consistent with protein-protein interaction events. Ten stacks per image were taken and a minimum of five fields per experiment were evaluated. The Blobfinder V3.2 free software (Centre for Image Analysis, Uppsala, Sweden) was used to quantify the number of blobs (or dots) present in each condition. For cell culture experiments, an average of blobs/cells of at least three independent experiments was performed and was normalized to the control condition. For paraffin embedded tumors, the specific number of blobs/cell was analysed. The respective negative controls were used in each experiment.

Intracellular protein interactions were analyzed using the Duolink^® kit (Olink Bioscience) which is based on the use of two unique and bi-functional probes called PLA™. Each probe consists of a secondary antibody attached to a unique synthetic oligonucleotide that acts as a reporter. Cells were cultured in chamber slides (Lab-Tek), fixed with 4% paraformaldehyde and permeabilized with TBS containing 0.1% Triton X-100. Non-specific binding was blocked with 1% bovine serum albumin/ 5% goat serum for 40 minutes. Slides were incubated with two primary antibodies overnight at 4°C. After washing, slides were incubated with the secondary oligonucleotide-linked antibodies provided in the kit. The oligonucleotides bound to the antibodies were hybridized, ligated, amplified, and detected using a fluorescent probe provided in the kit and nuclei were stained with DAPI. Slides were covered with fluorescent mounting medium and analyzed with a fluorescence microscope (Axio Imager).