Fig. 5

SUMOylation of hSSB1 is required for recruitment of NBS1 to DNA damage sites and enhances the binding affinity of hSSB1 to NBS1. a HeLa cells with stable knockout of hSSB1 by sgRNA were reintroduced with pTETON-hSSB1-WT, pTETON-hSSB1-DM, or an empty vector virus, as described in “Materials and methods,” and then the cells were lysed, and the proteins were measured by western blotting. b The cells in a were exposed to 10 Gy of gamma radiation followed by recovery for 2 h, fixed with paraformaldehyde solution and stained using the indicated antibody and DAPI; the scale bar represents 5 μm. c HeLa cells transfected with FLAG-hSSB1-WT or FLAG-hSSB1-DM were treated with laser microirradiation, and then stained with the indicated antibody and DAPI; the scale bar represents 5 μm. d HEK293T cells transfected with the indicated plasmids were treated 24 h later with or without 100 μM etoposide for 24 h, and then the cells were lysed and analyzed by western blotting or IP using the anti-FLAG antibody followed by western blotting. e HEK293T cells were transfected with the indicated FLAG-hSSB1 plasmid and the HA-SUMO3 plasmid for 24 h, and then treated with etoposide (100 μM) and ML-792 at the indicated concentrations for 24 h. Then, the cells were lysed and analyzed by western blotting or IP using the anti-FLAG antibody followed by western blotting. f Schematic illustration of FLAG-hSSB1-DM fused with 1–3 SUMO3 molecules. g HEK293T cells transfected with the indicated plasmids for 48 h were subjected to western blotting. h HEK293T cells transfected with the indicated plasmids for 24 h were treated with or without 100 μM etoposide for 24 h, and then the cells were lysed and analyzed by western blotting or IP using the anti-FLAG antibody followed by western blotting