Otub1 interacts with c-Maf and inhibits its K48-linked polyubiquitination. (A) An AP-MS assay was performed against c-Maf. From the c-Maf immunoprecipitates, Otub1 was identified with 6 unique peptides as underlined. (B) c-Maf and Otub1 plasmids were cotransfected into HEK293T cells; 48 hours later, cell lysates were prepared for IP and IB assays. TCL, total cell lysate. (C) MM cell line RPMI-8226 cells were infected with lentiviral Otub1, followed by IP/IB assays as indicated. (D) Cell lysates from RPMI-8226 and MM1.S cells were subjected to IP/IB with a c-Maf or Otub1 antibody or immunoglobulin G (IgG), followed by IB assays. (E) Purified c-Maf and Otub1 were incubated with E1, E2, Ub, and adenosine triphosphate (ATP) in a microtube. The proteins were then subjected to IB assays. (F-H) RPMI-8226 and MM1.S cells were infected with lentiviral Otub1, followed by IP/IB assays to examine the ubiquitination type. EV, empty vector. (I) RPMI-8226 and MM1.S cells were infected with lentiviral sgOtub1 to knock out Otub1, followed by IP/IB assays to determine c-Maf ubiquitination levels. (J) c-Maf, HERC4, Otub1, and Ub plasmids were cotransfected to HEK293T cells, followed by IP/IB assays.