Ub binding ability and nuclear localization of RNF168. A) Schematic representation of RNF168: RF (RING finger), MIU (Motif Interacting with Ubiquitin), NLS (Nuclear Localization Sequence). B) In vitro pull-down assay was performed using the indicated GST-tagged RNF168 constructs. GST-fusion proteins were incubated with 293T cell lysates (left panel) or synthetic polyUb2–7 linked by K48 (right panel) and analysed by SDS-PAGE. Immunoblot (IB) was performed on PVDF membrane treated as described in Methods and immunostained with antibodies directed against Ub and GST. C) 24 hours after transfection with the indicated FLAG-tagged RNF168 constructs, HeLa cells were immunostained with antibodies directed to FLAG. The nucleus was stained with To-Pro 3.