HERC4 is required for c-Maf ubiquitination. (A) HEK293 cells were transfected with an HA-c-Maf with or without a Flag-HERC4 plasmid. Forty-eight hours later, cells were treated with MG132 for 2 hours, followed by lysate preparation and co-IP with an anti-Ub–specific antibody and IB with indicated antibodies. (B) MM1.S cells were infected with HERC4 lentivirus. Ninety-six hours later, cell lysates were prepared for IP with an anti–c-Maf antibody followed by IB against indicated antibodies. (C) HA-c-Maf and Flag-HERC4 proteins were separately isolated and purified from overexpressing HEK293 cells with an anti-HA and Anti-Flag antibody, respectively. The purified proteins were then incubated in a 1.5-mL tube containing ATP and HA-Ub with or without E1-E2 mixture. Two hours later, the reaction was terminated and subjected to IP with an anti-Flag antibody followed by an anti–c-Maf antibody. (D) HEK293 cells were transfected with c-Maf, HERC4, wild-type (WT) or K48R or K63R mutated ubiquitin (Ub) plasmids. Twenty-four hours later, cells lysates were subjected to IP with an anti-HA antibody followed by IB with an anti-Flag antibody. (E) HEK293 cells were infected with individual HERC4 shRNAs; 48 hours later, cell lysates were prepared for evaluation of HERC4 protein levels by IB. (F) c-Maf, HERC4, and or HERC4 shRNA (#1) were introduced into HEK293 cells; 48 hours later, cell lysates were prepared for IP (with a Ub antibody) and IB against c-Maf. Cells were treated with MG132 before cell lysis.