Fig. 3

TRIP6 activates NF-κB signaling through interaction with TRAF6 and regulation of TRAF6 oligomerization and autoubiquitination. a A luciferase reporter assay was used to measure NF-κB reporter activity in TRIP6-transfected RAW264.7 cells treated with LPS (10 ng/ml). b, c TRIP6 interacts with TRAF6. Immunoprecipitation (IP) and immunoblot (IB) analyses of cell lysates of THP-1 cells (b) and HEK293T cells transfected with Myc–TRIP6 and Flag–TRAF6 (c). d LPS enhances the interaction between TRIP6 and TRAF6. IP and IB analyses of cell lysates of THP-1 cells treated with LPS (50 ng/ml). e, f TRIP6 promotes the oligomerization and autoubiquitination of TRAF6. HEK293T cells were transfected with the indicated plasmids for 30 h, the lysates were analyzed by IP with anti-GFP antibody (e) or anti-Flag antibody (f), and the oligomerization and autoubiquitination of TRAF6 were examined via Western blotting. g Interactions between TRIP6 and TRAF6 were analyzed by IP in intestinal samples from wild-type mice treated with or without DSS. h IP of endogenous TRAF6 from colon lysates of DSS-treated TRIP6^+/+ and TRIP6^−/− mice was performed, followed by the IB analysis of TRAF6 autoubiquitination using an anti-ubiquitin antibody