Fig. 3: USP44 ubiquitinates and degrades Ku80 by recruiting TRIM25.

a SDS-PAGE of HA-immunoprecipitated proteins separated from SUNE1 cells stably overexpressing HA-USP44. Red lines indicate the proteins of interest. b Co-IP with anti-HA or anti-FLAG antibody in SUNE1 cells revealed the exogenous association of USP44 and Ku80. c Immunofluorescence staining revealed the cellular location of exogenous HA-USP44 (green) and endogenous Ku80 (red) at 0.5 h after exposure to 6Gy IR. Scale bars, 10 μm. d USP44 inhibited Ku80 protein expression but not its mRNA expression in a dose-dependent manner. e The effect of CHX treatment and greyscale analysis of the results in 293T cells transfected with FLAG-Ku80 and HA-USP44 or the empty vector plasmids, as well as in sgNC or sgUSP44 SUNE1 cells. f The effect of MG132 and CQ treatment in 293T cells transfected with the indicated plasmids. g HEK293T cells transfected with FLAG-Ku80, HA-Ub and HA-USP44 or the empty plasmids were subjected to denature-IP and immunoblotted with the indicated antibodies. h Co-IP assay detecting the exogenous association of USP44 and TRIM25 and the endogenous association of USP44, TRIM25 and Ku80 in NPC cells. i TRIM25 inhibited Ku80 protein expression but not its mRNA expression in a dose-dependent manner. j The effect of MG132 and CQ treatment in 293T cells transfected with FLAG-Ku80 and FLAG-TRIM25 or the empty vector plasmids. k The effect of CHX treatment and greyscale analysis of the results in 293T cells transfected with FLAG-Ku80 and MYC-TRIM25 or the empty vector plasmids, as well as in sgNC or sgTRIM25 SUNE1 cells. l, m HEK293T cells transfected with the indicated plasmids or siRNAs were subjected to denature-IP and then immunoblotted with the indicated antibody. Data in d, e and i, k 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.