FIG. 6.

NOX2 controls IKK activity in RSV infection. (A) Control (Ctrl)- and NOX2-specific siRNA were transfected into A549. siRNA-transfected A549 were further infected with RSV at an MOI of 3 for the indicated times (hpi). WCE were resolved by SDS-PAGE and analyzed by immunoblotting (IB) as described in Fig. 5A. Efficiency of NOX2 downregulation was controlled by IB with anti-NOX2 antibodies. Equal loading was ensured using anti-tubulin antibodies. (B) WCE of siCtrl- and siNOX2-transfected A549 uninfected or infected with RSV for 6 hpi were subjected to an in vitro kinase assay as a substrate as described in Materials and Methods using immunoprecipitated IKK complex as a kinase and recombinant IκBα(1-55)-GST fusion protein as a substrate. The substrate was stained with Coomassie blue, and the IKK activity was measured by quantifying the incorporation of radioactivity (³²P) normalized to the amount of immunoprecipitated IKKβ detected by immunoblotting (IB). The results of a representative experiment are presented. The quantifications are expressed as the mean ± the SEM of four experiments (**, P < 0.01).