Dna ligase 4

A total lysate of 10–30μg protein was loaded on SDS-Gels and transferred to nitrocellulose membranes. The membranes were incubated with primary antibodies overnight and subsequently, probed with secondary HRP-conjugated antibodies. Primary antibodies: GAPDH (Life Science H86504M), Beta-Actin (Cell Signaling 4967), Ku70 (Santa Cruz sc-1486), Ku80 (Santa Cruz sc-9034), XLF (Santa Cruz sc-393844), DNA Ligase IV (Santa Cruz sc-28232), H2A.X (Cell Signaling 2595), γ-H2A.X-S139 (Cell Signaling 9718), Chk1 (Santa Cruz sc-7898), p-Chk1-Ser345 (Santa Cruz sc-17922), p-Chk1-S317 (Cell Signaling 2344), Chk2 (Santa Cruz sc-9064), p-Chk2-Thr68 (Santa Cruz sc-16297).

A total lysate of 30 μg protein was loaded on SDS-Gels and transferred to nitrocellulose membranes. The membranes were incubated with primary antibodies overnight and, subsequently, probed with secondary HRP-conjugated antibodies. Primary antibodies: GAPDH (Meridian Bioscience H86504M, Cincinnati, OH, USA), Ku70 (Santa Cruz Biotechnology sc-1486, Heidelberg, Germany), Ku80 (Santa Cruz Biotechnology sc-9034, Heidelberg, Germany), DNA ligase IV (Santa Cruz Biotechnology sc-28232, Heidelberg, Germany), Mre11 (Cell Signaling 4895, Danvers, MA, USA), DNA ligase III (BD Transduction Laboratories 611876, Franklin Lakes, NJ, USA), PARP (Cell Signaling 9542), DNA polymerase theta (Abcam ab80906, Cambridge, UK).

Whole tissues or MEFs single-cell suspensions were washed in PBS. Cells were lysed in 50–200 μL of 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% TritonX100, 1X phosphatase inhibitor cocktail 2 and 3 (ThermoFisher), and 1X protease inhibitor cocktail EDTA free (Roche) on ice for 45 min. Proteins migrated within Tris-Acrylamide gels and Tris-Glycine-SDS buffer and were transferred on PVDF membranes (Immobilon). Membranes were stained with the following antibodies: β-actin (mouse monoclonal AM4302, ThermoFisher), XRCC4 (goat monoclonal C-20, Santa Cruz), and DNA ligase IV (mouse monoclonal D-8, Santa Cruz). Membranes were revealed with appropriate secondary antibodies conjugated with infrared 700 or 800 dyes (LiCor). Immunoblotting was revealed by LiCor CLx analyzer and analyses were performed on Image Studio Lite software.