Journal: Nature communications

Article Title: The TDRD3-USP9X complex and MIB1 regulate TOP3B homeostasis and prevent deleterious TOP3B cleavage complexes.

doi: 10.1038/s41467-023-43151-z

Figure Lengend Snippet: Fig. 3 | USP9X deubiquitylates and protects TOP3B from proteasomal degra- dation. 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 indepen- dent 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 trans- fected 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

Article Snippet: The primary antibodies were diluted in 5% milk in PBST by 1:1000 for Mouse monoclonal anti-FLAG M2 (Millipore Sigma, St. Louis, MO, CAT#: F1804), 1:10000 for Rabbit monoclonal anti-GAPDH (Cell Signaling Technology, Danvers, MA, CAT#: 2118 S), 1:1000 for Rabbit monoclonal anti-HA (Cell Signaling Technology, Danvers, MA, CAT#: 3724 S), 1:1000 for Rabbit monoclonal anti-TDRD3 (Cell Signaling Technology, Danvers, MA, CAT#: 5942 S), 1:1000 for Mouse monoclonal anti-MIB1 antibody (B9, Santa Cruz Biotechnology, Santa Cruz, CA, CAT#: sc-393811), 1:1000 for Rabbit monoclonal anti-TOP3B antibody (abcam, Waltham, MA, CAT#: ab183520), 1:1000 for Rabbit monoclonal anti-USP9X antibody (Cell Signaling Technology, Danvers, MA, CAT#: 14898 S), 1:2000 for Mouse monoclonal anti-phospho (S139)-H2AX (JBW301, Millipore Sigma, St. Louis, MO, CAT#: 05-636), 1:1000 for Mouse monoclonal Anti-PAR PolymerMonoclonal Antibody (R&D Systems, Inc. a Bio-Techne Brand, CAT#: 4335-MC-100), 1:1000 for Ub (P4D1) antibody (Cat# sc-8017, Santa Cruz Biotechnology), 1:1000 for Rabbit polyclonal TRIM41 antibody, (Cat# ab111580, Abcam).

Techniques: Transfection, Western Blot, Quantitation Assay, Inhibition, Ubiquitin Proteomics, Construct