RNF168 has E3 Ub ligase activity and promotes the formation of K63-linked poly-Ub chains. A) The intrinsic Ub ligase activity of purified GST-RNF168 was analysed by in vitro ubiquitination assay as described in Methods section. Reaction mixtures were resolved by SDS-PAGE and analysed by immunoblotting as shown. The band present at lower molecular weight is likely a degradation product. (◀) indicates the size of GST-RNF168. B) HeLa cells transfected with cDNA encoding the GFP-tagged version of RNF168 and its RF-defective mutant (RF*) were immunostained using antibodies that recognized ubiquitinated proteins (FK2). C) HeLa cells were transfected with FLAG-RNF168 and co-immunostained with antibodies directed to FLAG (red) and to ubiquitinated proteins (green). In particular, we used the antibodies Apu2.07 and Apu3.A8, which specifically recognized K48- or K63-linked polyUb chains, in addition to antibodies FK2.