Fig. 2

UBE2O mediates c-Maf ubiquitination independent of an E3 ligase. a Flag-tagged ubiquitin (Ub), HA-tagged c-Maf, and Myc-tagged UBE2O were co-transfected into HEK293T cells for 48 h. Cell lysates were precipitated with anti-Flag beads followed by immunoblotting with an anti-c-Maf antibody. Total cell lysate (TCL) was used as a control with specific antibodies as indicated. b, c Myeloma cell lines LP1 (b) and KMS11 (c) were infected with lentiviral UBE2O, respectively, for 72 h, followed by cell lysate preparation and IP assay as indicated. d Purified c-Maf protein was incubated with E1, UBCH5c (E2), ATP, and Ub in the presence or absence of UBE2O protein at 37 °C for 2 h. After the termination, the reaction mixture was subjected to SDS-PAGE and immunoblotting assay against c-Maf. e, f The Ub mutants were co-transfected with UBE2O and c-Maf plasmids. Twenty-four hours later, cell lysates were prepared for immuunoprecipitation and immunoblotting assay