Otub1 stabilizes c-Maf and promotes its transcriptional activity. (A) Otub1 was cotransfected with c-Maf and its lysine-free variant (K0) in HEK293T cells; 48 hours later, cell lysates were prepared for IB assay as indicated. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. (B) Otub1 plasmids were cotransfected with c-Maf into HEK293T cells for the indicated periods, followed by IB assays. (C) MM cell lines RPMI-8226 and LP1 were infected with lentiviral Otub1 for 72 hours, followed by an IB assay. MOI, multiplicity of infection. (D) MM cell lines were transfected with siOtub1 for 48 hours, followed by MG132 treatment (8 hours) before being collected for IB assays. (E) c-Maf was cotransfected with Otub1 for 24 hours, followed by CHX treatment for the indicated periods and IB assays. (F) RPMI-8226 cells were infected with lentiviral Otub1, followed by CHX treatment and IB assays for endogenous c-Maf stability. (G) Otub1, MARE.Luci, c-Maf, and β-galactosidase (β-gal) plasmids were cotransfected into HEK293T cells; 24 hours later, cell lysates were prepared for luciferase (Luci.) activity measurement and IB assays. RLU, relative light units. (H) MARE.Luci, Otub1, and β-gal were cotransfected into RPMI-8226 cells for 48 hours, followed by luciferase and IB assays. (I-J) MM cell lines RPMI-8226 and MM1.S were infected with lentiviral Otub1 (I) for 72 hours or were transfected with siOtub1 or scramble (Scr) for 48 hours (J), followed by IB assays to evaluate the expression levels of c-Maf-targeted genes.