FIG 5.

Analysis of IKKε putative cleavage by DENV protease NS2B/3 and IKKε functionality after DENV infection. (A) Representation of IKKε structure, including two putative target sites for NS2B/3 protease. (B and C) IKKε cleavage evaluation by Western blotting. (B) 293/TLR3 cells were transfected with Flag-IKKε- and NS2B/3- or NS2B/3-S135A-encoding plasmids for 48 h, and then the cell lysate (20 μg) was analyzed using an anti-Flag or anti-IKKε antibody. (C) Mock- or DENV-infected cells after 24 h postinfection analyzed for IKKε endogenous expression in 40 μg of whole-cell extract using an anti-IKKε antibody. (D) IKKε functionality evaluation. In the top blot, the cells were transfected with IKKε for 24 h, and then infected with DENV-2 for another 24 h to evaluate the ability of DENV to affect IKKε-associated phosphorylation of S386-IRF3. (E) Densitometric analysis of pS386-IRF3 protein levels. The results were normalized to GAPDH. The asterisks represent a significant fold change compared to IKKε-transfected cells using one-way ANOVA: *, P < 0.05; **, P < 0.01 (n = 2). Each bar represents the mean ± SEM.