Figure 3. Knockdown of NOX2 expression impairs SeV-induced C-terminal IRF-3 phosphorylation and dimerization in A549 and human primary NHBE.
(A and B), A549 were pretreated with 3mM Tempol or the corresponding vehicle for 1h. A549 cells (C and D) or NHBE cells (E) were transfected with control (CTRL) or NOX2 RNAi. (A–E) Cells were left uninfected or infected with SeV (10 HAU/106 cells) for the indicated times. (A, C and E) WCE were analyzed by SDS-PAGE followed by immunoblot (IB) using anti-IRF3-Ser396 (IRF-3-P-Ser396) and anti-IRF3-Ser398 (IRF-3-P-Ser398) phosphospecific antibodies, anti-IRF-3 and anti SeV (the nucleocapsid N is shown) antibodies. Equal loading was controlled using anti-actin antibodies. (B and D), WCE analyzed in A and C were also resolved by native gel electrophoresis and revealed by immunoblot using anti-IRF3-Ser386 phosphospecific (IRF-3-P-Ser386) and anti-IRF-3 antibodies. M: monomer, D: dimer. Representative immunoblots of three different experiments are shown. hpi: hours post infection.