Fig 6. NEMO is activated through the binding of ubiquitin chains synthetized by TRAFs.

(A-C) WT, NEMO^−/− and NEMO^−/− THP-1 cells reconstituted with mCherry-tagged NEMO or mCherry-tagged NEMO-Mut (Y³⁰⁸S/D³¹¹N/H⁴¹³A/C⁴¹⁷A) were infected with SeV for the indicated times. Type I-IFNs, IL-6 and TNF were determined by bioassay or ELISA (A). The indicated gene induction was analyzed by RT-PCR (B). Cell lysates were analyzed by Western blot to detect the phosphorylation and expression of the indicated proteins (C). (D) 293T cells stably expressing Flag-tagged NEMO or Flag-tagged NEMO-Mut were infected with SeV for the indicated times and 293T cells were transiently transfected with Flag-TRAF6 for 24 h. Cell lysates were immunoprecipitated with the anti-Flag antibody. Half of the precipitates were subjected to a second-round immunoprecipitation. The precipitates were analyzed by Western blot with the anti-ubiquitin and ant-Flag antibodies (Upper panels). The whole cell lysates (WCL) were analyzed with the anti-Flag antibody to detect the expression of Flag-tagged NEMO and TRAF6 (Bottom). (E) to (F) The same experiment was performed as in (D) except that 293T MAVS^−/− cells stably expressing Flag-NEMO or Flag-NEMO-Mut (E) or TRAFs-deficient cells reconstituted with TRAF2, 3, or 6 or TRAF2/3/6-dR stably expressing Flag-tagged NEMO (F) were used. The precipitates were analyzed by Western blot with the anti-ubiquitin (Top) and ant-Flag (Bottom) antibodies. Data from (A) represent mean ± SD. Similar results were obtained in 3 independent experiments.