Fig. 5.

Effect of IRF3 and RelA knockdown on the IIR. A549 cells were transfected with RelA siRNA, IRF3 siRNA, and non-targeting control siRNA for 64 h. Then the cells were treated with ds-RNA for varying times. The cytoplasmic and nuclear proteins were extracted and subjected to SID-SRM-MS analysis. The temporal profiles of cytoplasmic RelA (A) and nuclear RelA (B) in the cells transfected with RelA siRNA or non-targeting control siRNA derived from SID-SRM-MS analysis are shown. The temporal profiles of cytoplasmic IRF3 (C) and nuclear IRF3 (D) in the cells transfected with IRF3 siRNA or non-targeting control siRNA derived from SID-SRM-MS analysis also are shown. Beta-actin was used to normalize between samples for SID-SRM-MS analysis. E, inhibition of expression of downstream target genes by RelA siRNA and IRF3 siRNA. Total RNA in RelA siRNA-transfected A549 cells was assayed for NFKBIA/IκBα and Groβ expression by Q-RT-PCR in response to ds-RNA (6 h). Non-targeting siRNA was used as a negative control (top panel). Total RNA in IRF3 siRNA transfected cells was assayed for ISG15 and IFNβ expression in response to ds-RNA (bottom panel).