Immunofluorescence and In Situ PLA Assays

For γH2AX immunofluorescence assays, cells were spun onto glass slides and stained as described (22 (link)). For USP36 immunofluorescence, cells were permeabilized in 0.5% Triton-X solution for 5 min at room temperature, and then fixed with 3% paraformaldehyde for 15 min. The samples were incubated with primary antibody for 1 h at 37°C, washed three times with TBST to remove the non-specific binding, incubated with the secondary antibody for about 30 min. Cells were then stained with DAPI to visualize nuclear DNA. The in situ PLA assay was carried out using a Duolink in situ PLA kit (Sigma, # DUO92101) according to the manufacturer's instructions. Cells grown on cover glass were washed with PBS twice and incubated in 3% paraformaldehyde for 15 min, and permeabilized in 0.5% Triton-X solution for 5 min at room temperature. After blocking, samples were incubated with USP36 and PrimPol antibodies for 1 h. Samples were washed twice times and incubated with Duolink PLA Probe for 1 h at 37°C, Duolink Ligation buffer for 30 min at 37°C and Amplification buffer for 100 min at 37°C. Cells were then stained with DAPI to visualize nuclear DNA. The coverslips were mounted onto glass slides with anti-fade solution and visualized using a Nikon eclipse 80i fluorescence microscope. Counting of the PLA signal dots was done using ImageJ.