Duolink detection kit

Cells were cultured on glass coverslips in 6-well plates to at least 80% confluence and fixed in ice-cold methanol for 20 min for both proximity ligation assays (PLA) E-cadherin/b-catenin and E-cadherin/p120. PLA was performed using Duolink Detection kit (Olink Bioscience, Sweden), according to the manufacturer’s instructions for Duolink Blocking solution and Detection protocol. Briefly, slides were blocked, incubated with antibodies directed against E-cadherin cytoplasmic domain (610,182, BD Biosciences or Clone 24E10, #3195, Cell Signaling, USA), b-catenin (C2206, Sigma-Aldrich, USA) and p120 (610,134, BD Biosciences, USA), followed by incubation with the secondary PLA probes (anti-mouse Minus and anti-rabbit Plus) conjugated to unique oligonucleotides. Amplification template oligonucleotides were hybridized to pairs of PLA probe and circularized by ligation. Rolling circle amplification was performed and detection of amplified DNA was possible by addition of complementary oligonucleotides labeled with Cy3 fluorophore. Coverslips were mounted on Vectashield with DAPI (Vector Laboratories, USA). Images were acquired on a Carl Zeiss Apotome Axiovert 200 M Fluorescence Microscope (× 20 and × 40 objectives; Carl Zeiss, Germany) with an Axiocam HRm camera and processed with the Zeiss Axion Vision 4.8 software. Quantification of PLA signals was achieved using BlobFinder V3.2.42.

Cells were fixed and incubated with anti-Kindlin-3 antibody (Genosphere), followed by Alexa 488 secondary antibody and examined with a laser-scanning confocal microscope (Leica-Lasertechnik, Heidelberg). In the negative controls the primary antibody was substituted with PBS.
In situ PLA used to assess protein-protein interactions, cells grown on Lab-tek chamber slides (Nunc, #154534), transfected with Kindlin-3 or control siRNA and immediately fixed were subjected to In situ PLA using the Duolink Detection kit (Olink Bioscience, Sweden) according to the manufacturer's instructions. Briefly, slides were blocked, incubated with antibodies directed against Kindlin-3 (Genosphere), Integrins β1, β3 or β5 (Abcam ab58524, Santa Cruz sc-14009, sc-14010) or Talin (Abcam ab11188) and thereafter incubated with PLA probes, which are secondary antibodies (anti-rabbit and anti-mouse) conjugated to unique oligonucleotides. Circularization and ligation of the oligonucleotides was followed by an amplification step. The products were detected by a complementary fluorescently labeled probe. Protein complexes were visualized in a laser-scanning confocal microscope (Leica-Lasertechnik) as bright fluorescent signals.

A proximity ligation assay (PLA) to detect an association between RCAS1 and ADAM9 was carried out using a Duolink detection kit (Olink Bioscience, Uppsala, Sweden). Briefly, SiSo, MCF-7, ADAM9 siRNA-transfected SiSo, and RCAS1 siRNA-transfected SiSo cells [20 (link)] were seeded into 8-well chamber slides. On the next day, cultures were fixed in 90% ethanol/5% acetic acid and subjected to PLA. Slides were incubated with mouse anti-RCAS1 (MBL) and rabbit anti-ADAM9 (Chemicon) antibodies and then secondary antibodies conjugated to unique DNA probes (PLA probe MINUS and PLUS) were added. Ligation and circularization of the DNA were followed by a rolling circle amplification step, and reactions were detected by a complementary Tex613 fluorophore-labeled DNA linker [21 (link)]. Slides were evaluated using an LSM 510 META confocal microscope (Zeiss, Jena, Germany).

Proximity ligation assay was performed to detect CLU and Cdc25C interaction using the CLU and Cdc25C antibodies. To visualize the bound antibody pairs, the Duolink Detection Kit (Duo92008) with PLA plus and minus probes for mouse and rabbit (Olink Bioscience) was used, according to the manufacturer's description. Cell slides were mounted with the Duolink Brightfield Mounting Medium (Olink Bioscience).

Cells were harvested, washed in PBS, and mounted onto slides by cytospin (Shandon Cytospin 3; Thermo Fisher) at 1,000 rpm for 5 min, were immediately fixed in 4% PFA (4% formaldehyde in PBS) on ice for 30 min and thereafter subjected to in situ PLA using Duolink Detection kit (Olink Bioscience, Uppsala, Sweden) according to the manufacturer's instructions for Duolink Blocking solution and Detection protocol. Briefly, slides were blocked, incubated with antibodies directed against mouse-anti EBNA1 (clone E1-2.5; Acris GmbH) and rabbit-anti Survivin (Novus biologicals; NB500-201) and thereafter incubated with PLA probes, which are secondary antibodies (anti-mouse and anti-rabbit) conjugated with oligonucleotides PLA probe minus and PLA probe Plus). Circularization and ligation of the oligonucleotides was followed by an amplification step. Slides were mounted using Vectashield (Vector Laboratories Inc, Burlingame, CA). Images were captured with a 63X lens on a Leica SP5 II Confocal microscope (Leica Microsystems) using LAS AF software for image processing and quantification. The number of foci, visualized as bright fluorescent signals, was counted in 10-15 cells/slide. n (number of slide) = 3
Additional Methods are provided in Supplemental Materials.