Fig. 6

ATM phosphorylates UFL1 and enhances its activity. a Flag-UFL1 was expressed in U2OS cells. Following vehicle or ATM inhibitor KU55933 treatment, the cells were irradiated with IR. Flag-UFL1 was immunoprecipitated and blots were probed with phospho-SQ/TQ antibody and Flag antibody. b Flag-UFL1 was expressed in atm+/+or atm−/− MEF cells. Cells were irradiated with IR and UFL1 phosphorylation was then examined as in a. c Flag-UFL1 or the S462A mutant was expressed in U2OS cells. Cells were treated with or without 10 Gy IR. Thirty minutes later, cells were lysed and incubated with Flag antibody-conjugated agarose beads. The immunoprecipitates were blotted with phospho-SQ/TQ antibody and Flag antibody. d Flag-tagged wildtype (WT) and S462A mutant UFL1 expressing cells were treated with or without 2 Gy IR and fixed and stained with indicated antibodies. Scale bars, 10 µm. e Flag-tagged UFL1 or S462A mutant expressing U2OS cells were irradiated with 10 Gy IR. Thirty minutes later, UFL1 WT and the S462A mutant were purified from the cells, and incubated with purified UBA5, UFC1, UFL1, UFM1, and H4 protein at 30 ^°C for 90 min. The reaction was assessed by probing the blot with the indicated antibody. f His-UFM1 was expressed in ATM proficient or deficient cells. His-UFM1 conjugated protein was purified under denaturing conditions. The purified samples were blotted with indicated antibodies. g Flag-tagged UFL1 or the S462A mutant was expressed in U2OS cells. Cells were harvested at indicated time points following IR and blotted with indicated antibodies. h Colony formation assay following IR was performed with UFL1-depleted cells reconstituted with WT UFL1 or the S462A mutant. The data is presented as mean±s.e.m. of n = 3 independent experiments. Dots depict individual data points. Statistical significance was calculated using two-way ANOVA. **P < 0.01. Source data are provided as a Source Data file