Figure 4.

8-HD inhibits the kinase activity of IKKε and interferes with the TRIF-mediated complex formation composed of TRAF3, TANK, and IKKε. (A) Immunoprecipitation and in-vitro kinase assay of p-IKKε as an enzyme and IRF-3 as a substrate. Immunoprecipitates of p-IKKε from LPS-induced RAW264.7 cells were incubated with immunoprecipitates of IRF-3 for 1 h in the presence of ATP (400 μM) with or without 8-HD (50 μM) as indicated. Phospho- and total levels of IRF-3 were then determined by immunoblotting. (B) The effect of 8-HD on the TRAF3 and IKKε interaction. Endogenous TRAF3 was immunoprecipitated from LPS-induced RAW264.7 cells pre-treated with or without 8-HD (50 μM). The level of phosphorylated IKKε was determined by immunoblotting. H.C: Heavy chain. (C) Treatment of 8-HD has a marginal effect on polyubiquitination. RAW264.7 cells (5 × 10⁶ cells/mL) were pre-incubated with MG132 (50 mM) for 4 h. The cells were then pre-treated with 8-HD (50 μM) for 30 min, followed by incubation with LPS (1 μg/mL) for 30 min. The level of polyubiquitination was analyzed by immunoblotting of whole cell lysates with ubiquitin (Ub) antibody. (D) RAW264.7 cells (2.5 × 10⁷ cell/mL) were grown overnight followed by 8-HD (50 μM) or DMSO treatment for 30 min and LPS (1 μg/mL) for 30 min afterward. CETSA was performed and the remaining soluble protein of IKKε was analyzed by immunoblotting analysis.