Duolink pla technology

PLA was conducted as previously described^{26 (link)} using Duolink PLA Technology (Merck). Briefly, samples were fixed, permeabilized, and incubated with RNAPII-S2P (Bethyl A300-654A) and PCNA (Santa Cruz Biotechnology sc-56) primary antibodies as described for IF assays. PCNA and RNAPII-S2P antibodies were used at 1:500 dilution. Then, samples were incubated with PLA-specific secondary antibodies and PLA reagents according to the manufacturer’s guidelines. Duolink in situ PLA probe anti-rabbit PLUS (Merck DUO92002), Duolink in situ PLA probe anti-mouse MINUS (Merck DUO92004) and Duolink-Detection Reagents Red (Merck) were used to perform the PLA reaction. Finally, nuclei were stained with DAPI, mounted in ProLong Gold AntiFade reagent (Invitrogen) and images acquired with a Leica DM6000 microscope equipped with a DFC390 camera (Leica) at ×63 magnification and LAS AX image acquisition software (Leica). PLA foci number per cell were quantified for all conditions.

Proximity ligation assays (PLAs) were performed using Duolink PLA technology (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. Briefly, cells were washed once with 1× PBS and treated with cytoskeletal (CSK) extraction buffer [0.2% Triton X-100, 20 mM HEPES-KOH (pH 7.9), 100 mM NaCl, 3 mM MgCl₂, 300 mM sucrose, 1 mM ethylenebis (oxyethylenenitrilo)-tetraacetic acid (EGTA)] containing 4% formaldehyde at 25 °C for 10 min. The cells were then washed three times with 1× PBS, permeabilized with 0.5% NP-40 in 1× PBS for 5 min, and blocked with 5% BSA in PBS at 25 °C for 1 h. After incubation with the indicated primary antibodies at 4 °C overnight, the cells were washed three times with 1× PBS and incubated with anti-mouse and anti-rabbit plus PLA probes (PLA kit, Sigma-Aldrich) at 37 °C for 1 h. The PLA reaction was performed using Duolink in Situ Detection Reagents (PLA kit) according to the manufacturer’s instructions. Finally, the cells were washed three times with buffer B, stained with 4′,6-diamidino-2-phenylindole (DAPI) during the second wash, and mounted with antifade mounting medium (Beyotime) on slides, coverslipped, and sealed with nail polish. Images were captured using a Zeiss LSM 800 fluorescence microscope (40×) and quantified using ImageJ software.

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)}.