Pla probe anti goat minus

The proximity ligation assay was performed using the Duolink In Situ Kit (Sigma) in accordance with the manufacturer’s instructions. Cells were seeded on 18 mm circular slides, washed with PBS twice, fixed with 4% PFA in PBS for 10 min and permeabilized with 0.2% Triton X-100 for 10 min. Cells were then blocked in 3% BSA, 0.1% Tween-20 in 4X SSC for 1 h at room temperature. Cells were then incubated with primary antibody overnight at 4°C [1:500 goat anti-RNA polymerase II antibody (PLA0292, Sigma) with 1:500 rabbit anti-PCNA antibody (PLA0079, Sigma)]. The next day cells were washed 2x in PBS and then incubated with pre-mixed PLA probe anti-goat minus and PLA probe anti-rabbit plus (Sigma) for 1 h at 37°C. The following ligation and amplification detections steps were followed identically to the Duolink In Situ Kit (sigma). Slides were then stained with DAPI and imaged on a LeicaDM18 microscope at ×100. Negative controls were treated identically but anti-RNA polymerase II antibody and PCNA antibody were omitted.

Cells were grown on coverslips, washed with PBS, fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.2% Triton X-100 for 5 min. Cells were then blocked in 3% BSA, 0.1% Tween-20 in 4XSSC for 1 h at room temperature. Cells were then incubated with primary antibody overnight at 4 °C [1:500 goat anti-RNA polymerase II antibody (PLA0292, Sigma) with 1:500 rabbit anti-PCNA antibody (PLA0079, Sigma)]. The next day after washing with 1 ×  PBS twice, cells were incubated with pre-mixed PLA probe anti-goat minus and PLA probe anti-rabbit plus (Sigma) for 1 h at 37 °C. The subsequent steps in proximal ligation assay were carried out with Duolink In Situ Kit (Sigma) in accordance to manufacturer’s instructions. In brief, cells were washed with Buffer A for 5 min three times, ligation reaction for 30 min at 37 °C, washed with Bufer A for 2 min two times, and Amplification for 100 min at 37 °C. After washing with Buffer B for 10 min three times and 0.01% Buffer B for 1 min, slides were then stained with DAPI and imaged on LeicaDM18 microscope at ×100. Negative controls were treated identically but anti-RNA polymerase II antibody was omitted.

NMCFs grew on coverslips. After washing three times with PBS, cells were fixed with 4% paraformaldehyde for 15 min, and then permeabilized with 0.2% Triton X-100 for 5 min. Cells were then blocked with 3% BSA, and 0.1% Tween-20 produced by SSC for 1 h at room temperature. Cells were then incubated with primary antibody against p53 (1:500; Cell Signaling Technology, USA, #2524), and against PML (1:500; Novus, USA, #NB100-59787) overnight at 4°C. Then cells were incubated with pre-mixed PLA probe anti-goat minus and PLA probe anti-rabbit plus (Sigma-Aldrich, St Louis, USA) for 1 h at 37°C. The proximal ligation assay was conducted with the Duolink In situ Kit (Sigma-Aldrich, St Louis, USA) according to the manufacturer's instructions. Nuclei were stained with DAPI and images were captured with a fluorescence confocal laser scanning microscope (Zeiss, Oberkochen, Germany).