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. 2009 Sep 28;206(10):2091–2099. doi: 10.1084/jem.20081761

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© 2009 Iwakiri et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jem.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 3. — EBER is detected as a complex with La in the culture supernatants and the complex is stimulatory for TLR3. (A) Detection of La, L22, and PKR in culture supernatants. Mutu+ cells (1 × 10⁶) were transfected with Flag-tagged La, L22, or PKR plasmid and cultured for 48 h. Flag-tagged La, L22, and PKR were detected by immunoblotting of the cell lysates (top) and immunoprecipitation of culture supernatants (bottom) using an anti-Flag antibody. Three or more independent experiments were performed for each assay. (B) Detection of EBER1 in the immunoprecipitates. RNA was extracted from the immunoprecipitates and subjected to 30 cycles of RT-PCR to detect EBER1. Three independent experiments were performed. (C) Effect of immunoprecipitates from culture supernatants on IFN-β induction. EBER-positive (EBER+) or EBER-knockout (EBER−) EBV-infected AGS cells were transfected with control (control) or Flag-tagged La plasmid (Flag La) and cultured for 48 h, followed by immunoprecipitation of culture supernatants using anti-Flag antibody. Both EBER− and EBER+ immunoprecipitates were added into the media of EBER-knockout EBV-infected AGS cells transfected with negative control siRNA (Flag La+nc) or TLR3 siRNA (Flag La+TLR3si), and IFN-β induction was analyzed by RT-PCR. The data presented are representative of three independent experiments.