Fig. 3. USP15 stabilizes TRIM25 by counteracting its ubiquitylation by LUBAC.
(A) HEK 293T cells were transfected with plasmid encoding TRIM25-FLAG together with empty plasmid or plasmid encoding USP15-V5, as well as a plasmid encoding a HA-tagged ubiquitin (Ub) mutant in which all of the lysines except Lys48 (K48) are mutated (HA-K48only-Ub). Thirty-six hours later, cells were treated with 50 μM MG132 for 6 hours. WCLs were subjected to immunoprecipitation with α-FLAG antibody, and samples were then analyzed by Western blotting with α-HA and α-FLAG antibodies. (B) HEK 293T cells were transfected with plasmid encoding TRIM25-FLAG with or without plasmids encoding HOIP-Myc and HOIL-1L-V5, as well as with empty plasmid or plasmids encoding V5-tagged USP15 WT or the USP15 C783A mutant. Mono- and polyubiquitylation of TRIM25 were determined by immunoprecipitation with α-FLAG antibody and Western blotting analysis with α-Ub antibody. The presence of USP15, HOIL-1L, HOIP, and actin was determined in WCLs by Western blotting analysis with α-V5, α-Myc, and α-actin antibodies. (C) Ectopically expressed USP15 enhances the stability of TRIM25. HeLa cells were transfected with empty plasmid or plasmid encoding Myc-tagged USP15. Forty hours later, cells were treated with 100 μM cycloheximide (CHX) for the indicated times. Total protein amounts in WCLs were determined by bicinchoninic acid (BCA) assay, and equivalent amounts of WCLs were then subjected to SDS–polyacrylamide gel electrophoresis (SDS-PAGE), followed by Western blotting analysis with α-TRIM25, α-Myc, and α-actin antibodies. Data in all panels are representative of at least three independent experiments. Data from the densitometric analysis of three experiments represented by the blots in (B) and (C) are presented in figs. S4 and S5, respectively.