FIGURE 3:

Ubiquitination of ECSIT is critical for the interaction with and translocation of p65 NF-κB to the nucleus. (A) HEK293-TLR4 cells were transfected with Flag-ECSIT and treated or not with 100 ng/ml LPS for 45 min. The cytosolic and nuclear fractions were prepared from the cells, and immunoblot (IB) with antibodies to anti-Flag, anti-p65, anti-PDI, and anti-PCNA was performed. (B) HEK293T cells were transfected with mock or Flag-ECSIT vector and immunoprecipitated (IP) with anti-Flag antibody, followed by IB with anti-Flag antibody. The immunoprecipitants were reacted in a K63-Ub reaction kit and treated with or without CYLD enzymes (left). The reactants were mixed with lysates prepared from HEK293T cells transfected with mock or p65- or p50-expressing vector, as indicated (right), and then the mixtures were centrifuged and washed three times with lysis buffer, and Western blotting with antibodies to anti-Flag, anti-p65, or anti-p50 was performed. (C) HEK293T cells were transiently transfected with Flag-ECSIT wt or Flag-ECSIT truncated mutants, along with the HA-Ub vector, as indicated, and then the IP assay was performed with anti-Flag antibodies, followed by IB with antibodies to anti-Flag or anti-Ub. (D) Comparison of sequences of wt ECSIT and K372A ECSIT mutant.