Fig. 7: IRGM1 is polyubiquitylated and degraded by CRL4B complex.

a Immunoprecipitation was performed to detect interactions between CUL4B, DDB1 and IRGM1. Protein extracted from small intestine of mice was immunoprecipitated with the indicated antibodies individually. b The effect of CUL4B on IRGM1 ubiquitination was confirmed by immunoprecipitation and detected by Western blot with indicated antibodies. IRGM1, HA-Ub expression constructions and siRNA (siCUL4B or siNC) were transfected into HEK293T cells as indicated in lane 1, 2, and 3. c In vitro ubiquitination of IRGM1 by the CRL4B complex. Purified His-IRGM1 was incubated with E1, E2, Ub and ATP in the absence and presence of the CRL4B complex (Flag-CUL4B IP products) as indicated in lane 1–4. Ubiquitination of His-IRGM1 was analyzed by Western blot using anti-Ub antibody. d Single lysine mutations on IRGM1 defect ubiquitination by CRL4B. Immunoprecipitation was performed with wildtype and mutant Irgm1 variants together with Flag-CUL4B, HA-Ub with treatment with MG132 for 3 h as indicated in lane 1–5. Western blot was performed and detected with indicated antibodies. Input (3%) was used for Western blotting. e Immunoprecipitation was performed to detect interactions between CUL4B and WDR77. Protein extracted from mice small intestine or HEK293T cells was immunoprecipitated with the indicated antibodies. f, g GST pull down assays identify the direct interaction among IRGM1, DDB1 and WDR77. GST-WDR77, His-DDB1 and His-IRGM1 were expressed in E. coli individually followed by protein purification and immunoprecipitated pull-down. The proteins were detected using indicated antibodies. Input (15%) was used for Western blot. h In vivo ubiquitination assay of IRGM1 by the CRL4B complex with or without WDR77 interfering.