Research Paper Volume 12, Issue 11 pp 10969—10982

P22077 inhibits LPS-induced inflammatory response by promoting K48-linked ubiquitination and degradation of TRAF6

Figure 5. P22077 promotes the degradation of TRAF6. (A) HEK293T cells were transfected with Flag-TRAF6 and HA-Ub plasmid for 24h, and then treated with or without P22077 (7.5 μM) for 6 h, the Flag-TRAF6 protein was detected by immunoblot. (B) Mouse peritoneal macrophages were pretreated with or without P22077 (7.5 μM) for 2 h, and then stimulated with LPS (100 ng/mL) for 1 and 2 h. Immunoblot assay was used to analyze TRAF6 protein. (C) Mouse peritoneal macrophages were pretreated with various dose of P22077 (0, 2.5, 5 and 7.5 μM) for 2 h, and then stimulated with LPS (100 ng/mL) for 1 h. Immunoblot assay was used to analyze TRAF6 protein. (D) Mouse peritoneal macrophages were treated with cycloheximide (100 μg/ml) in the absence or presence of P22077 (7.5 μM) for 0, 6 and 12 h. Immunoblot assay was used to analyze TRAF6 protein. (E) Mouse peritoneal macrophages were pretreated with P22077 (7.5 μM), MG132 (20 μM), Chloroquine (50μM) or 3-MA (1 mM) for 2 h, and then stimulated with LPS (100 ng/mL) for 1 h. Immunoblot assay was used to analyze TRAF6 protein. Numbers below lanes indicated densitometry of the protein presented relative to β-actin. Similar results were obtained from three independent experiments and data were presented of one representative experiment.