FIG 4.

DENV protease interacts with human IKKε. (A) 293/TLR3 cells were transfected with HA-NS2B/3 or HA-NS2B/3-S135A and Flag-IKKε or empty plasmid and at 48 h posttransfection were evaluated by proximity ligation assay (PLA). Single detection of proteins as controls for expression: IRF3 (a), IKKε (b), HA-NS2B/3 (c), and HA-NS2B/3-S135A (d). Red fluorescence indicates positive expression of these proteins and blue indicates nuclear staining by DAPI. (e to j) Double detection of protein-protein interactions. HA-tagged proteins were stained with a primary anti-HA antibody and a secondary PLA Minus probe, and IKKε was stained using a primary anti-IKKε antibody and a secondary PLA Plus probe. When the proteins are in close proximity, fluorescence is generated. The absence of red fluorescence reflects no protein-protein interaction. Shown are the endogenous expression of IKKε (e and f) and IKKε overexpression (g and h). (B) Cells were lysed at 48 h posttransfection and extracts coimmunoprecipitated with antibody against HA, and the bound and unbound fractions were analyzed by immunoblotting for IKKε.