Poly(I:C) stimulation induces TLR3 phosphorylation, translocation of IRF3 into the nucleus, and increased expression of primary response genes (IFNβ, ISG15, IP10, RANTES) in BMMCs. BMMCs were stimulated with 10 μg/mL poly(I:C) for different time intervals as indicated. (A) TLR3 protein was precipitated from protein lysates of activated BMMCs using anti-TLR3 antibodies and subjected to 10% SDS-PAGE. Blots were analyzed by anti–p-Tyr antibodies (top blot). To prove that equal amounts of TLR3 protein were loaded in each sample, blots were stripped and reprobed with anti-TLR3 antibodies (bottom blot). Stimulation of BMMCs with 100 ng/mL LPS was used as negative control. Position of TLR3 is indicated on the right, and molecular mass is indicated on the left. IP indicates immunoprecipitation; WB, Western blotting. (B) Nuclear extracts from BMMCs were analyzed in 10% SDS-PAGE with anti-IRF3 antibodies (top blot). For loading control, expression of Myc protein was detected on the same membrane after stripping (bottom blot). (C) BMMCs generated from 2 mice were stimulated with 10 μg/mL poly(I:C) for 1 hour. Total RNA was extracted from cells, reverse transcribed, and subjected to PCR amplification using specific primers for IFN-β, ISG15, IP10, and RANTES as indicated. The amount of cDNA was equalized by PCR amplification of β-actin. A mock PCR (no DNA) was included as a negative control. The data represent 3 separate experiments with comparable results.