Fig. 3: USP9X deubiquitylates and protects TOP3B from proteasomal degradation. | Nature Communications

Fig. 3: USP9X deubiquitylates and protects TOP3B from proteasomal degradation.

From: The TDRD3-USP9X complex and MIB1 regulate TOP3B homeostasis and prevent deleterious TOP3B cleavage complexes

Fig. 3

a Loss of USP9X destabilizes both TOP3B and TDRD3 proteins in HEK293 cells. b, c Depletion of USP9X decreases the half-lives of both TDRD3 and TOP3B in HEK293 cells. Cells were transfected with USP9X siRNA and treated with CHX (10 μg/ mL) for the indicated time. Panel b is a representative Western blot and panel c the quantitation of TOP3B and TDRD3 protein levels from three independent experiments. Data are plotted as means ± standard deviations (SD). d Inhibition of USP9X with the DUB inhibitor Degrasyn (WP1130) decreases both TOP3B and TDRD3 protein levels in HEK293 cells. Cells were treated with Degrasyn (WP1130; 2 μM, 24 h), pan-DUB inhibitor PR619 (5 μM, 24 h) or transiently transfected with TDRD3 siRNA. e USP9X can deubiquitylate TOP3B and TDRD3-USP9X axis is important for the deubiquitylation process. HEK293 cells were transfected with HA-Ubiquitin construct and siTDRD3, siUSP9X or both (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, TDRD3, and USP9X antibodies (GAPDH as loading control for input samples). The figure is representative of three independent experiments. f Treatment with the proteasome inhibitor MG132 rescues the TOP3B protein levels in USP9X-depleted cells. HEK293 cells were transfected with USP9X siRNA for 48 h. Before harvest, cells were treated with MG132 (1 µM, 24 h) as indicated and lysates were subjected to western blotting with USP9X, TOP3B, and TDRD3 antibodies (GAPDH loading control). g USP9X downregulation does not affect TOP3B protein level in TDRD3KO HCT116 cells. After transfecting the cells with USP9X siRNA for 48 h, lysates were subjected to Western blotting with USP9X and TOP3B antibodies (GAPDH as loading control). h Ectopic expression of USP9X increases both TOP3B and TDRD3 protein levels. After transfection of HEK293 cells with USP9X-FLAG construct for 48 h, cell lysates were subjected to western blotting with TOP3B, TDRD3, and USP9X antibodies (GAPDH as loading control). Source data are provided as a Source Data file.

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