Fig. 4: USP44 deubiquitinates and stabilises TRIM25 to promote Ku80 ubiquitination.

a USP44 promoted Ku80 protein expression but not its mRNA expression in a dose-dependent manner. b, c The effect of CHX (b), MG132 and CQ (c) treatment in 293T cells transfected with FLAG-TRIM25 and HA-USP44 or the empty vector plasmids, as well as in sgNC or sgUSP44 SUNE1 cells. d, e HEK293T cells transfected with HA-USP44 or the empty vector (d) and sgNC or sgUSP44 SUNE1 cells (e) co-transfected with FLAG-TRIM25 or MYC-TRIM25 and a vector encoding HA-WT-Ub or its mutants (HA-K48O-Ub or HA-K63O-Ub) were subjected to denature-IP and immunoblotted with the indicated antibodies. f HEK293T and NPC cells transfected with vector plasmid, HA-USP44 or HA-USP44 (C282A) were immunoblotted with the indicated antibodies. g HONE1 cells transfected with the vector plasmid, HA-USP44 or HA-USP44 (C282A) together with MYC-TRIM25 and HA-K48O-Ub were subjected to denature-IP and immunoblotted with the indicated antibodies. h Mass spectrometry analysis of TRIM25 ubiquitination sites. i HEK293T cells were transfected with the vector plasmid or HA-USP44, HA-Ub and Flag-TRIM25 WT or KR mutants, subjected to denature-IP with anti-Flag beads and then analysed by immunoblot with an anti-HA or anti-Flag antibody. j SUNE1 and HONE1 cells exposed to IR (6Gy) transfected with the indicated plasmids and siRNAs were fixed 0.5 h later and co-immunostained with the anti-Ku80 antibody. Scale bars, 10 μm. Data in a and b are presented as the mean ± SD; the P values were determined using the two-tailed Student’s t-test); n = 3 independent experiments. Source data are provided as a Source Data file.