FIGURE 5:

K372 ubiquitination of ECSIT is functionally involved in activating NF-κB induced by LPS stimulation. (A) NF-κB reporter assay in HEK293-TLR4 cells transfected with mock, different concentrations of wt ECSIT, or K372A mutant ECSIT in the presence or absence of LPS. All error bars represent SDs of the mean from triplicate samples. (B, C) p65 or p50 DNA–binding assay in HEK293-TLR4 cells transfected with the indicated vectors, mock, wt ECSIT, or K372A mutant ECSIT, in the presence or absence of LPS. All error bars represent SDs of the mean from triplicate samples. (D) THP-1 cells were infected with lentiviral particles containing control lentiviral particles or shRNA-targeted human ECSIT lentiviral particles according to the manufacturer's protocol. Control THP-1 (Ctrl) or ECSIT-knockdown THP-1 (ECSIT^KD THP-1) were cultured in puromycin-containing medium (4 μg/ml) for 2 wk to select stable clones, and immunoblots (IB) with antibodies to anti-ECSIT or anti-GAPDH were performed to evaluate knockdown efficacy. (E) NF-κB reporter assay in control (ctrl) or ECSIT^KD THP-1 cells transfected with mock, different concentrations of wt ECSIT, or K372A mutant ECSIT in the presence or absence of LPS. All error bars represent SDs of the mean from triplicate samples. (F, G) p65 or p50 DNA–binding assay in ctrl or ECSIT^KD THP-1 cells transfected with the indicated vectors, mock, wt ECSIT, or K372A mutant ECSIT in the presence or absence of LPS. All error bars represent SDs of the mean from triplicate samples. (H, I) Ctrl or ECSIT^KD THP-1 cells were transfected with mock, wt ECSIT, or K372A mutant ECSIT, treated with or without LPS, and the levels of IL-6 (H) and IL-1β (I) were measured by ELISA. All error bars represent SDs of the mean from triplicate samples. *p < 0.05 and **p < 0.01.