Usp28 counteracts the ubiquitination of Nbs1 to stabilize the MRN complex and to restore double-strand break (DSB) response in RecQL4-depleted cells.A, localization of Usp28 at DSB sites in U2OS cells, 1 h after neocarzinostatin (NCS) or mock treatment. The scale bar represents 10 μm. B, ubiquitination of Nbs1 in cells expressing wt or C171A mutant Usp28, 1 h after NCS treatment. C, schematic diagram of truncated Usp28 proteins and their interaction with Nbs1. D, expression of Nbs1 interaction domains in Usp28 inhibits the restoration of DSB responses by Usp28 overexpression. Percentages of Mre11-, phospho-ATM-, or Rad51 foci-positive cells transfected with the indicated proteins. Data in graphs are means ± SD; n = 3. ∗∗∗p < 0.001, ∗∗p < 0.01. CAT, catalytic domain; UBA, ubiquitin-associated domain; UIM, ubiquitin-interaction motif.