HERC4 regulates c-Maf degradation. (A) NIH3T3 cells expressing c-Maf and the cyclin D2 promoter–driving luciferase were transfected with a selected subset of E3 ligase plasmids. Twenty-four hours later, cyclin D2 transactivation was assessed by the luciferase assay. (B) NIH3T3 cells were cotransfected with c-Maf and E3 candidates for 48 hours, and cell lysates were then prepared for IB to measure c-Maf protein level. 1, pcDNA3.1; 2, pcDNA3.1; 3, HERC4; 4, Itch; 5, CDKN1B; 6, COPS8; 7, CXCR4; 8, EDN1; 9, FBXL14; 10, WWP2; 11, HIF1A; 12, pcDNA 3.1. (C) NIH3T3 cells were transfected with HERC4 and several HECT-containing E3 plasmids for 48 hours before cell lysate preparation for c-Maf protein determination with IB. 1, pcDNA3.1; 2, pcDNA3.1; 3, HERC4; 4, HACE1; 5, FLJ21156; 6, UREB1; 7, DKFZP564G092 (HERC4); 8, ITCH; 9, WWP2; 10, ZNF313; 11.TRIM8; 12. RNF111; 13. HERC4-myc. (D) HEK293 cells were transfected with c-Maf, HERC4, or vector control. Forty-eight hours later, cells were treated with CHX (100 μg/mL) for 0, 0.5, 1, 2, 3, 4, 6, and 12 hours to inhibit de novo protein synthesis and were then harvested for IB. (E) HEK293 cells were transfected with MafA, MafB, or c-Maf along with increased HERC4. Forty-eight hours later, cells were harvested for IB to determine the Maf protein levels.