RNF168 ubiquitinates histone H2AX and is recruited to DDR foci upon DNA damage. A) In vivo ubiquitination of histone H2AX was evaluated in 293T cells co-transfected with GFP alone, GFP-RNF168 WT or RF* together with FLAG-tagged histone H2AX. Lysates were treated as described in Figure 4B. Immunoblot with anti-FLAG antibodies revealed the presence of higher molecular weight proteins compatible with mono- (◆), di- (◆◆) and tri- (◆◆◆) ubiquitinated forms of the histone. Low and high exposure of the immunoblot are shown. B) Cells expressing FLAG-RNF168 were treated with etoposide (5 μM, 1 hour) prior to fixation. To mark DNA damage foci, IF staining was performed using antibodies directed to the phosphorylated form of histone H2AX (γH2AX), to the consensus sequence phosphorylated by the DDR kinases ATM and DNA-PK (P-S/TQ) and the mediator protein 53BP1. C) IF analysis was performed on HeLa cells transfected with FLAG-RNF168 and treated with increasing doses of etoposide, as shown. Immunostaining with anti-γH2AX indicated the number and position of DDR foci. D) DNA damage was induced using different genotoxic agents, such as etoposide (5 μM), UV radiation (UV, 10 J/m2) and Hydroxyurea (HU, 2.5 mM). IF analysis was performed as in C. E) HeLa cells transfected with GFP-RNF168, GFP-RF* or GFP-MIU1-2* were treated with etoposide (5 μM) for 1 hr prior to fixation. IF staining performed with antibodies anti-γH2AX revealed the differences in the recruitment at DDR foci of different constructs. F) HeLa cells transfected as in E were incubated with etoposide (5 μM) for 1 hr. To highlight the retention of different constructs in the detergent-insoluble compartments after DNA damage induction, cells were pre-treated (TX+) or not (TX-) with Triton X-100 before fixation. DDR foci are marked by anti-γH2AX immunostaining.