Decreased stability of the IRF8K108E mutant. (A) RAW macrophages stably expressing HA-tagged K108 or E108 variant were treated with CHX for the indicated periods of time (hours) and for up to 6 hours. Cell lysates were subjected to SDS-PAGE and immunoblotted with a mouse anti-HA mAb. Actin was used as an internal loading control. (B) RAW macrophages stably expressing HA-tagged K108 or E108 were treated with CHX for 6 hours and 12 hours with or without the proteasome inhibitor MG132 (10 µM), and cell lysates were analyzed by immunoblotting. (C) Relative protein expression was assessed by densitometry using ImageJ. Results are represented as means ± SD of 3 independent experiments. (D) HEK 293T cells were transiently transfected with K108 or E108 variant, along with ubiquitin, and 24 hours later, cells were treated with MG132. IRF8 was immunoprecipitated from cell lysates followed by immunoblotting with anti-ubiquitin antibody. IRF8 was also detected by immunoblotting from total cell lysates. The left and right panels come from the same experiment, membrane and film, and thus can be used for comparison. (E) HEK 293T cells were transiently transfected with K108 or E108 variant, with or without V5-tagged SUMO3 and/or HA-tagged SENP1. Cell lysates were immunoprecipitated with anti-IRF8 antibody and immunoblotted with anti-V5 antibody. Total cell extracts were also immunoblotted with anti-V5 antibody to show SUMOylation of other proteins and SENP1 de-SUMOylating activity.
