Electrophoretic Mobility Shift Assay

Human Ku70/His-Ku80 was purified from baculovirus-infected insect cells (26) and human APLF and XRCC4 were expressed in bacteria as previously described (12,13). Unlabeled and 5’ FAM labeled RNA and DNA probes were RNase-free synthesized by Integrated DNA Technologies (IDT) (Supplementary Table S1D). Recombinant proteins were incubated with the labeled probes in 1x EMSA binding buffer (25 mM HEPES-KOH pH 7.5, 50 mM NaCl, 1 mM DTT, 1 mM EDTA, 10 % glycerol) supplemented with 0.1 μM ultrapure BSA (Invitrogen, AM2616) in a final volume of 20 μl. Samples were incubated at room temperature for 25–30 min in the dark. Then samples were loaded in non-denaturing 5 % acrylamide gels and run in 1x EMSA running buffer (50 mM Tris, 380 mM glycine, 2 mM EDTA) at 100V for 45 min in the dark. After that, gels were imaged using the ImageQuant LAS-4000 (Fujitsu Life Sciences) or a Chemidoc MP Imaging System (Bio-Rad).