Expression of TLR3 in murine MCs. (A) BMMCs from 2 mice were left unstimulated or were stimulated for 1 hour with 10 μg/mL poly(I:C). RT-PCR was performed as described in “Materials and methods.” The amplified products were electrophoresed on 1.5% agarose gel. A mock PCR (without cDNA) was included to exclude contamination. The amount of cDNA analyzed was similar in different samples, as shown by PCR amplification of β-actin cDNA. (B) BMMCs were treated with 10 μg/mL poly(I:C) for 24 hours (pIC) or left untreated. Expression of TLR3 protein was analyzed by Western blotting in cell lysates using specific anti-TLR3 antibodies. Position of TLR3 is indicated on the right, and molecular mass is indicated on the left. Expression of TLR3 was shown by surface staining of BMMCs (C) or PMCs (E) as described in “Materials and methods.” Intracellular staining of BMMCs (D) or PMCs (F) for TLR3 was performed after fixation and permeabilization of cells. For surface staining of BMMCs, anti-TLR3 antibodies and biotinylated secondary anti–mouse IgG₁ antibodies and APC-labeled streptavidin were used. For surface staining of PMCs and intracellular staining, A647-labeled anti-TLR3 antibodies were used. Gray and white areas show staining with anti-TLR3 and isotype-matched control antibodies, respectively. Results are representative of 4 independent experiments.