RNF169 is dispensable for ubiquitin-dependent assembly of repair factors at DSB-modified chromatin. (A) U2OS cells transfected with the indicated siRNAs for 72 h were subjected or not subjected to IR (4 Gy), fixed 1 h later, and coimmunostained with 53BP1 and MDC1 or γ-H2AX antibodies. Bars, 10 µm. (B) Immunoblot analysis of the experiment in A. The asterisk denotes a cross-reactive band. (C) HEK293T cells were transfected with the indicated combinations of plasmids for 24 h. To analyze histone H2A ubiquitylation, FLAG immunoprecipitates were immunoblotted with the Myc antibody. (D) Ubiquitylation reactions containing recombinant H2A, HA-ubiquitin, E1, and E2 (UbcH5a) were incubated in the presence or absence of purified His₆-RNF168 or His₆-RNF169 for 1 h and processed for immunoblotting with HA, H2A, and His₆ antibodies. (E) Reactions containing the indicated components were processed as in D and immunoblotted with the HA antibody to visualize RNF168 and RNF169 autoubiquitylation. CTRL, control; IP, immunoprecipitation; MM, molecular mass; Ub, ubiquitin; WCE, whole-cell extract; WB, Western blot.