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. 2022 Feb 25;29(9):1730–1743. doi: 10.1038/s41418-022-00960-x

Fig. 2. OTUD6A deubiquitinates and stabilizes c-Myc.

Fig. 2

A A schematic diagram to show the screening of potential DUB candidates that might play oncogenic role in PrCa. Amplified genes were marked in bold, and potential oncogenic DUB candidates in red-bold. B OTUB2, OTUD6A, and OTUD7B, but not other OTU family members interacted with c-Myc. Immunoblot of Flag-immunoprecipitants and whole-cell lysis (WCL) derived from HEK293T cells transfected with haemagglutinin (HA)-c-Myc and Flag-tagged OTU DUB family members. C Endogenous OTUD6A binds with c-Myc protein. Immunoblot of c-Myc-immunoprecipitants derived from 22Rv1 cells. D OTUD6A, but not OTUB2 or OTUD7B, deubiquitinated c-Myc. Immunoblot of WCL and nickel-nitrilotriacetic acid (Ni-NTA) pull-downs from HEK293T cells transfected with HA-c-Myc, His-ubiquitin (His-Ub) and Flag-tagged constructs. Cells were pretreated with 10 µM of MG132 for 6 h before harvesting. E Catalytic-dead mutant of OTUD6A (OTUD6A-C152A) failed to deubiquitinate c-Myc oncoprotein. Immunoblot of WCL and Ni-NTA pull-downs from HEK293T cells transfected with HA-c-Myc, His-Ub and Flag-tagged OTUD6A or the catalytic-dead mutant OTUD6A-C152A. Cells were pretreated with 10 µM of MG132 for 6 h before harvesting. F, G Ectopic WT, but not the C152A mutant form of OTUD6A, extended the protein half-life of c-Myc in 22Rv1 cells. Immunoblot (F) and quantification (G) of cell lysis derived from 22Rv1 cells transient transfected with either empty vector (EV), OTUD6A-WT or OTUD6A-C152A, and treated with 200 µg/ml of cycloheximide (CHX) for indicated time. The protein abundances were in G. The relevant raw data are provided in Supplementary Materials.