Fig. 1

UFL1 protein accumulates at DSBs through the MRN complex. a U2OS cells stably expressing UFL1 Tet-on shRNA were treated with doxycycline (Dox) for 3–5 days, and then treated with or without 2 Gy IR. After 30 min, cells were harvested and lysed with NETN buffer. Cell lysates were incubated with UFL1 antibody + Benzonase. The immunoprecipitates were blotted with indicated antibodies. b Immunofluorescence of UFL1 and γH2AX in U2OS cells irradiated with IR (0.5 Gy). c Triamcinolone acetonide (TA) treatment induces the translocation of RFP-I-SceI-GR fusion protein from the cytoplasm to the nucleus and generates one double strand break at the cutting site. The protein localization was detected by indicated antibodies. d UFL1 foci formation at different time points following 0.5 Gy IR treatment. e, f UFL1 foci formation was analyzed in Mre11 knockdown cells or NBS1 deficient cells (NBST). g NBST cells were transfected with Vector (V) or Flag-NBS1 and treated with or without 2 Gy IR. After 30 min, the cells were lysed and immunoprecipitation with UFL1 antibody with Benzonase treatment was performed. The immunoprecipitates were blotted with indicated antibodies. h The schematic diagram of NBS1 protein domain. i U2OS cells were treated with or without 2 Gy IR, and the cell lysates were pulled down with GST, GST-NBS1 FHA+BRCT (1+2) proteins. After washes, the beads were boiled and analyzed with indicated antibodies. Scale bars, 10 µm. Source data are provided as a Source Data file