Figure 1.

The tyrosine kinase c-Src is activated by dsRNA, associates with TLR3 and is required for dsRNA-induced IRF-3 and STAT-1 activation. (A) Human mDCs were incubated with dsRNA (100 μg/ml) and activation of c-Src was analyzed by immunoblotting (IB) with an antibody specific to activated phospho-Src. (B) HEK cells expressing Flag-tagged TLR3 were stimulated with dsRNA (100 μg/ml) and lysates were immunoprecipitated (IP) with anti-Flag or mouse IgG and immunoblotted (IB) for the indicated proteins. (C) HEK293 cells were transiently cotransfected with plasmids encoding TLR3 and a luciferase reporter gene containing the Gal4 upstream activation sequence, and expression vectors for Gal4-DBD or Gal4-IRF-3. After 24 h, cells were treated with PP2, PP3, or SU6656 before stimulation with dsRNA. (D) Nuclear extracts were prepared from dsRNA-treated SYF and c-Src-expressing control cells and analyzed by immunoblotting (IB) with an IRF-3 antibody. The blots were reprobed with the nuclear protein XRCC1 as a loading control. (E, F) Determination of IRF-3 localization in c-Src-, Yes-, and Fyn-deficient cells and c-Src-expressing control cells (c-Src) by confocal microscopy. (E) Cells were treated or not with dsRNA and stained intracellularly for IRF-3 (Alexa546). (F) Average percentages of IRF-3 nuclear translocation in SYF cells and c-Src-expressing control cells with nuclear IRF-3 as assessed by confocal microscopy. A total of 200 cells were counted under the different conditions. (G, H) DsRNA-elicited or IFN-β-induced activation of STAT-1 in SYF and c-Src-expressing cells was determined by immunoblotting with an antibody specific to activated STAT-1 (Y⁷⁰¹) and STAT-1. (I) HEK293 cells were transiently cotransfected with vectors encoding TRIF, 0.5, 2, or 20 ng of kinase-inactive c-Src (K297R), and a luciferase reporter gene for IFN-β. Luciferase reporter gene activity was measured after 24 h.