Duolink 2 detection kit

Detection of an interaction between proteins was assessed using the Duolink II Detection Kit (Sigma-Aldrich) according to the manufacturer specifications. The signal was visualized as a distinct fluorescent spot and was captured on an Olympus BX-51 upright Fluorescence Microscope using a ×60/1.35 oil lens. The number of PLA signals in a cell was quantified in Image J using a Maximum Entropy Threshold and Particle Analysis where 50 cells in each treatment group were analyzed for at least three independent experiments. RGB images were converted to black/white images with the Invert LUT from Image J.

PLA was performed using Duolink II Detection Kit to confirm protein–protein interactions according to the manufacturer’s instructions (Sigma–Aldrich). Briefly, blocked cells were incubated with anti-CD147 and anti-CD276 antibodies for 1 h on wet ice. After washing with PBS, the lipid rafts of cells were stained with 10 ug/mL of Cholera Toxin Subunit B (CT-B) for 30 min at 4 °C. The cells were fixed with 4% paraformaldehyde for 20 min at room temperature and then incubated with PLA probes (oligonucleotides) for 1 h at 37 °C. After washing with PBS, cells were ligated for 30 min at 37 °C. Subsequent amplification with polymerase was performed for 150 min at 37 °C. Nuclei were stained with DAPI. Red fluorescent signals from the PLA were visualized using a confocal laser scanning microscope (LSM 880; Carl Zeiss, Jena, Germany). The number of red dots of each cell was quantified using Image J software (NIH, Bethesda, MD, USA), where 100 cells of each group were analyzed in at least three independent experiments.