Fig. 2: Enhanced ubiquitylation of TOP3B in the absence of TDRD3 and recruitment of USP9X by TDRD3.

a Enhanced TOP3B ubiquitylation in the absence of TDRD3 in HEK293 cells. Cells were transfected with HA-Ubiquitin construct and TDRD3 siRNA (as indicated) for 48 h and subjected to TOP3B pulldown. Pulled down and input samples were resolved on SDS-PAGE and probed with Ubiquitin, TOP3B and TDRD3 antibodies. The figure is representative of three independent experiments. b Enhanced ubiquitylation of TOP3B in TDRD3KO HCT116 cells. HCT116 wild-type and TDRD3KO cells were subjected to TOP3B pulldown. Pulled down and input samples were resolved on SDS-PAGE and probed with Ubiquitin, TOP3B and TDRD3 antibodies. The figure is representative of three independent experiments. c Ectopic expression of TDRD3 decreases TOP3B ubiquitylation in TDRD3KO HCT116 cells. TDRD3KO HCT116 cells were transfected with TDRD3-FLAG constructs for 48 h and subjected to TOP3B pull down. Pulled down and input samples were resolved on SDS-PAGE and probed with Ubiquitin, TOP3B and TDRD3 antibodies. The figure is representative of three independent experiments. d TOP3B pulldown-LC-MS/MS showing that endogenous TOP3B interacts with USP9X and TDRD3 in HEK293 cells. Shown are the number of identified peptide spectral matches (PSM). e HA-tag pulldown-LC-MS/MS showing TOP3B interaction with USP9X in HEK293 cells. After transfection of HA-TOP3B, HEK293 cells were subjected to HA-tag pulldown followed by LC-MS. f, g USP9X pulldown-Western blot experiments showing that depletion of TDRD3 suppresses TOP3B-USP9X interaction in HEK293 cells. Panel f is a representative Western blot and panel g is the quantitation of three independent experiments. Data are plotted as means ± standard deviations (SD). Two tailed unpaired t-test. *P value = 0.002. h USP9X pulldown-Western blot experiments showing the absence of TOP3B-USP9X interaction in TDRD3KO HCT116 cells. Source data are provided as a Source Data file.