Poly 1

The agarose beads (Sigma) conjugated to poly(C) were washed several times in 50 mM Tris (pH 7.0)–200 mM NaCl and then resuspended in 50 mM Tris (pH 7.0)–50 mM NaCl. To make poly(I-C)-coated agarose beads, poly(C)-coated beads were resuspended in two volumes of 2 mg of poly(I) (Sigma)/ml prepared in 50 mM Tris (pH 7.0)–150 mM NaCl. The mixture was then rocked gently overnight at 4°C, collected by centrifugation at 1,000g, washed with 50 mM Tris (pH 7.0)–150 mM NaCl, and resuspended in the same buffer as a 50% final slurry. For poly(C) and poly(I-C) pull-down assays, poly(C)- or poly(I-C)-coated beads were equilibrated in binding buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1% NP-40) as a 10% slurry and then combined with an equal volume of whole-cell extract that was prediluted to contain 300 ug of protein. The cell extracts from primary MEF cells or HEK293 cells were supplemented with protease and phosphatase inhibitors and 25 U of RNase inhibitor/ml. The mixture was incubated with gentle agitation for one hour at 4°C. Beads were centrifuged at 1,000g, rinsed three times with binding buffer, and then resuspended in three volumes of 1x SDS-PAGE sample buffer. Samples were incubated at 95°C for five minutes, centrifuged at 13,000g for 30 seconds, and loaded immediately onto SDS-PAGE gels and processed for immunoblot analysis.