Otub1 deubiquitinates c-Maf in both canonical and noncanonical manners. (A) A skeptical illustration of Otub1 domains and key residues in its catalytic activity. (B) c-Maf, Ub, and Otub1 variants were cotransfected into HEK293T cells followed by IP/IB assays as indicated. (C) WT, K71R, or C91S Otub1 variants were cotransfected into HEK293T cells with c-Maf plasmids for 24 hours, followed by CHX treatment and IB assays. (D) The density of c-Maf bands from panel C was analyzed. (E) Lentivirus containing Otub1 and its mutants were infected into MM cell lines for 96 hours, followed by IP/IB assays to evaluate c-Maf polyubiquitination levels. EV, empty virus. (F) Otub1 and USP5 or USP7 plasmids were cotransfected into HEK293T cells for 24 hours, followed by IB assays. (G-H) WT or ΔN Otub1 cells were transfected into HEK293T cells alone or together with UBE2O (G) or UBE2D3 (H) for 24 hours, followed by IP/IB assays as indicated.