Fig. 7: Irgm1 protects Keap1 from ubiquitination and degradation.

A Up: protein-protein docking simulation of Irgm1 (palecyan) and Keap1 (lightpink). Down: the key amino acids from Irgm1 (green) and Keap1 (pink) in binding. Yellow dashed lines indicate the hydrogen bonds. B Immunoprecipitation showed the interaction between endogenous Irgm1, Keap1, and Usp25 in RAW264.7 cells. C Co-IP analysis of the interaction between endogenous Irgm1, Keap1, and Usp25 in HEK293T cells transfected with FLAG-IRGM and HA-KEAP1. D Co-IP analysis of the interaction between Irgm1 and Keap1 in BMDMs induced with or without 40 ng/ml RANKL for 24 h. E Mapping the domains in IRGM that interact with KEAP1. FLAG-tagged domain 1 (D1) and D2 of IRGM were expressed in HEK293T cells and immunoprecipitated. The immunoprecipitates were then analyzed for the presence of KEAP1. F WB showed Keap1 expression in RAW264.7. Cells were pretreated with 5 μM MG132 or 20 μM CQ or 1 mM 3-MA for 2 h and then stimulated with 40 ng/ml RANKL for 24 h. G Quantitative results from F were normalized to GAPDH and presented as means ± SD (n = 3). ^∗p < 0.05, ^∗∗p < 0.01, and ns (not statistically significant) vs the indicated groups. H, I WB showed Keap1 levels in RAW264.7 under different situations. Cells transfected with Irgm1 knockdown lentivirus (H), or overexpression lentivirus (I) were treated with 50 ng/ml cycloheximide (CHX) for the indicated times to measure the half-lives of keap1. Quantitative data were normalized to GAPDH and presented as means ± SD (n = 3). J, K WB showed the ubiquitination expression of endogenous Keap1 and the expression of Usp25 in RAW264.7 cells depleted of Irgm1 (J) or in RAW264.7 cells overexpressing Irgm1 (K). Cells were treated with 5 μM MG132 and 40 ng/ml RANKL for 6 h.