Duo92007

An in situ proximity ligation assay (PLA, Sigma, DUO92007) was performed as previously described 30 (link). Briefly, AsPC-1 and BxPC-3 cells were seeded in a 12-well chamber overnight, fixed for 30 mins at room temperature, and then permeabilized with 0.1% Triton-X100 for 2 mins. After incubation with DuoLink blocking buffer for 60 min at 37 ℃, the cells were incubated with primary antibodies against PRLR and NEK9 from two different species at 4 ℃ overnight. Next, the cells were incubated with DuoLink PLA MINUS and PLUS Probes at 37 ℃ for 1 hr, and then ligation solution was added to form a closed circle at 37 ℃ for 30 mins. Next, the amplification reaction was performed by polymerase at 37 ℃ for 100 mins. Finally, the cells were stained with DAPI and mounted. All the assays were performed under humidified conditions to avoid false-positive results.
Additional protocols and procedures are described in the supplementary materials and methods.

The PLA was performed as described previously (36 ). Briefly, pCAGGS-Flag, pCAGGS-Flag-BIRC6-236aa, or pCAGGS-CypD was transfected into IPEC-J2 cells. Cells were then infected for 12 h with 1 MOI TGEV and fixed immediately with 4% paraformaldehyde for 15 min at RT. Cells were permeabilized with 0.01% Tween 20/PBS for 10 min at RT, incubated with a mixture of two primary antibodies (mouse anti-Flag and rabbit anti-VDAC1) followed by an oligonucleotide-linked secondary antibody, and then exposed to polymerase and nucleotides (DUO92007, Sigma–Aldrich). Nuclei were counterstained with DAPI. Images were captured under a laser scanning confocal microscope.

Cells were cultured on coverslips, rinsed with DUO link wash Buffer B according to the manufacturer’s protocol (DUO92007, Sigma-Aldrich, USA). Then they were incubated in blocking solution for 30 min at 37 °C, and the appropriate primary antibody was applied overnight at 4 °C. Next, cells were exposed to secondary antibodies conjugated to oligo-nucleotide iPLA probes (cat DUO92001, cat DUO92005, MINUS and PLUS, Sigma-Aldrich) for 1 h at 37 °C. Ligase activity performed coupled with rolling circle amplification and the products were hybridized by oligo-nucleotide probes labeled with a fluorophore. Red punctuate signals were captured using confocal scanning microscopy.