Requirement of NOX2 and Reactive Oxygen Species for Efficient RIG-I-Mediated Antiviral Response through Regulation of MAVS Expression
Figure 4
NOX2 is essential for IKKε expression and SeV-induced TBK1 activity.
A549 were pretreated with the vehicle or the indicated concentrations of DPI (10–30µM) (A and B) or transfected with CTRL or NOX2 RNAi (C and D). Cells were then left uninfected or infected with SeV (40 HAU/106 cells) and harvested at different hours post infections (hpi). (A and C), WCE were resolved by SDS-PAGE and analyzed by immunoblot (IB) using anti-TBK1, anti-IKKε and anti-actin antibodies. (B and D), TBK1 activity was monitored by in vitro kinase assay using GST-IRF-3-(aa387-427). Reactions were resolved by SDS-PAGE and IRF-3 substrate was detected by coomassie blue (CB) staining and radioactivity incorporation (32P). TBK1 activity was expressed as the ratio of radioactivity incorporation over the amount of immunoprecipitated kinase detected by immunoblot (IB) and quantified by densitometric analysis. Results are expressed as percentage of the activity measured after SeV infection in respective control cells. In B, black bars correspond to vehicle-treated cells, white bars corresponds to DPI-treated cells. In D, black bars correspond to CTRL RNAi-tranfected cells and white bars correspond to NOX2 RNAi-transfected cells.(**, p<0.01; ***, p<0.001; mean ± SEM of at least three independent experiments)