Quantification and Immunodetection of DNA Repair Proteins

For the quantification and inmunodetection of specialized GFP tagged Pol κ and Pol η, 53BP1 and γH2AX respectively, cells were fixed in 2% paraformaldehyde (PFA)/2% sucrose and permeablized with 0.1% Triton X-100 in phosphate buffered saline (PBS) as described previously (Mansilla et al., 2013 (link)). For the detection of chromatin-bound protein ice cold, 0.5% Triton CSK buffer was used (10 mM Pipes, pH 7.5, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2). Proteins were preextracted 1 min or 5 min when detecting p21 or RPA/PCNA respectively. EdU was detected following manufacturer’s instructions (Click-iT EdU kit– C10338 Invitrogen). Blocking was performed overnight in PBS 2% donkey serum (Sigma, St. Louis, Missouri). Coverslips were incubated for 1 hr in primary antibodies: 53BP1(Santa Cruz, Dallas, Texas), γH2AX (EMD Millipore, Massachusetts), p21 (c-19 Santa Cruz), RPA (NA18 EMD Millipore), PCNA (Abcam), pH3 (ser10 Millipore). Secondary anti-mouse/rabbit-conjugated Cy2/Cy3 antibodies were from Jackson Immuno Research and anti-rabbit alexa 488 from Invitrogen. GFP-tagged specialized Y polymerases and GFP-PCNA were detected by GFP autofluorescence. Nuclei were stained with DAPI (SIGMA). Images were obtained with a Zeiss Axioplan confocal microscope or a Zeiss Axio Imager A2.