Duolink in situ pla kit

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.

Dissociated commissural neurons were stimulated with BSA or Netrin-1 for 2 or 5 min and fixed with 4% PFA in PBS. The samples were blocked with 10% BSA [immunoglobulin G (IgG) free] and 0.1% Triton X-100 in PBS (pH 7.4) for 1 hour at room temperature and then incubated with antibodies against DCC (1:50) (Santa Cruz Biotechnology sc-6535) and ARHGEF7 (1:50) (MilliporeSigma, 07-1450-I) or GIT1 (1:50) (Novus Bio NBP2-22423), diluted in PBS with 1% BSA (IgG free) and 0.1% Triton X-100, overnight at 4°C. The proximity ligation reaction was performed with the Duolink in situ PLA kit (Sigma-Aldrich) according to the manufacturer’s instructions.

At 70% confluence, TPC1 cells were seeded on glass cover slips (Thermo Fisher), cultured overnight in a 12-well plate, and treated with CDDP for 12 hours. Thereafter, TPC1 cells were fixed with 4% paraformaldehyde in PBS for 10 min, permeabilized with 0.1% Triton X-100 in PBS for 5 min, and incubated with 5% BSA for 1 hour at room temperature. Cells were incubated with rabbit anti-MUL1 (1:100) and mouse anti-AKT (1:100) antibodies at 4°C overnight. Cells were incubated with anti-rabbit PLUS and anti-mouse MINUS PLA probes in Duolink in situ PLA kit (Sigma-Aldrich), according to the manufacturers’ protocol. After nuclei were stained using Hoechest (Thermo Fisher), cells were observed with a confocal laser microscope (Nikon, Tokyo, Japan).