a HEK293FT cells were transfected with HA-IDO1, Flag-Ubiquitin, or MYC-USP14 as indicated. Polyubiquitination of IDO1 was then examined by immunoprecipitation with HA-beads and analyzed. Cells were treated with MG132 (10 µм) for 6 h before being harvested. b HCT116 cells treated with IU1 (50 µм) for 24 h or the cells stably transfected with control shRNA and USP14 shRNA were treated with MG132 (10 µм) for 6 h before being harvested. Lysates were immunoprecipitated with anti-IDO1 and analyzed. c HEK293FT cells were transfected with HA-K48-Ub and Flag-IDO1 with or without MYC-USP14 as indicated and cultured for 48 h. Cells were immunoprecipitated with anti-Flag and analyzed. d HCT116 cells were immunoprecipitated with anti-IDO1 or anti-TRIM21 and analyzed. e Flag-Ubiquitin was transfected together with HA-TRIM21 WT or HA-TRIM21 LD mutant in HEK293FT cells. Cells were treated with MG132 (10 µм) for 6 h before being harvested, then immunoprecipitated with anti-Flag and analyzed. f HCT116 cells with stable expression of control shRNA and TRIM21 shRNA were infected with Flag-IDO1. Cells were treated with MG132 (10 µм) for 6 h before being harvested, then immunoprecipitated with anti-Flag and analyzed. g Purified Flag-IDO1 was incubated with IgG control, purified HA-TRIM21 WT, or HA-TRIM21 LD mutant in the ubiquitination assay mix, and the sample was probed for the indicated proteins. h HA-Ub (WT, K48R, and K63R mutants) was transfected with or without Flag-TRIM21 in HEK293FT cells. Cells were immunoprecipitated with anti-HA and analyzed. i HCT116 cells were transfected with the indicated constructs and analyzed. j HEK293FT cells were transfected with IDO1, TRIM21, and Flag-tagged USP14 WT, C114A, or ΔUBL for 48 h and analyzed. k HA-TRIM21 was transfected with Flag-USP14 WT, C114A, or ΔUBL in HCT116 cells. Cells were treated with MG132 (10 µм) for 6 h before being harvested, then immunoprecipitated with anti-IDO1 and analyzed. The experiment was repeated three times independently with similar results (a–k). Source data are provided as a Source Data file.