In situ PLA for Protein-Protein Interactions

For detection of protein-protein interactions, in situ PLA was performed. The components used (Duolink PLA Technology, Sigma-Aldrich) were as follows: anti-mouse PLA plus probe, anti-rabbit PLA minus probe, anti-goat PLA minus probe and Detection Reagents Orange. HUVEC were grown on chamber slides (Ibidi^® μ-Chamber 12 well on glass slides, Martinsried, Germany) till they reached 80% confluence. Following fixation and blocking, the cells were incubated with the primary antibodies: mouse anti-RPTPβ/ζ (1:250, #610180, BD Biosciences), rabbit anti-CDK5 (1:100 in TBS-T; #sc-173, Santa Cruz Biotechnology), mouse anti-CDK5 (1:100 in TBS-T; #H00001020-M01A, Abnova, Taipei, Taiwan), goat anti-p35 (1:100 in TBS-T; #sc-31102, Santa Cruz Biotechnology). Subsequently, the cells were incubated with secondary antibodies conjugated with oligonucleotides, after hybridization and ligation of which, the DNA was amplified resulting in red fluorescence signals. Nuclei were counterstained with Draq5; cells were mounted with Mowiol 4–88 and visualized with Leica SP5 confocal microscope. Estimation of nuclei and cytoplasm size was performed using the Duolink ImageTool software. To calculate the total number of spots per cell, an algorithmic procedure was used as previously described^{55 (link)}.