FIG. 5.

IRF7 is a substrate for TRAF6 E3 ligase and identification of IRF7 lysine sites for ubiquitination. (A) IRF7 is a substrate for TRAF6 E3 ligase. His-TRAF6 and His-TRAF6(C70A) proteins were expressed in and purified from E. coli, and Flag-IRF7 protein was expressed in and purified from 293T cells. An in vitro ubiquitination assay was performed. After the reaction, solutions were heated in 1% SDS at 95°C to dissociate protein interactions before IP with Flag antibody. Western blotting (WB) was performed with the antibodies indicated. IB, immunoblotting; αFlag, anti-Flag. (B) Identification of IRF7 ubiquitination sites. GST-IRF7-Flag and GST-TRAF6 were purified from E. coli. An in vitro ubiquitination assay was performed (see details in Materials and Methods). Samples were separated on SDS gels and immunoblotted with Ub antibody. Blots were stripped for reprobing IRF7 with Flag antibody. WT, wild type.