Stimulation of MCs with NDV induces TLR3 phosphorylation, c-Kit down-regulation, chemokine production, and up-regulation of costimulatory molecules. Stimulation of BMMCs with NDV (2.4 HA/mL) for indicated time points leads to TLR3 phosphorylation (A). TLR3 protein was precipitated from protein lysates 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). Position of TLR3 is indicated on the right, and molecular mass is indicated on the left. (B) Transcription of primary response genes in BMMCs is induced after stimulation with NDV. BMMCs were stimulated with NDV for 1 hour in concentrations as indicated. Total RNA was extracted from cells, reverse transcribed, and subjected to PCR amplification using specific primers for IFN-β, ISG15, IP10, and RANTES. 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 are representative from 2 separate experiments with comparable results. (C) Stimulation of BMMCs with NDV (2.4 HA/mL) for 24 hours leads to down-regulation of c-Kit expression (open histogram) compared to unstimulated control (gray shaded histogram). Dotted histogram shows staining with isotype-matched control antibodies. Results are representative of 4 independent experiments. Productions of MIP-1β (D) and RANTES (E) were analyzed in supernatants of BMMCs in response to virus infection. BMMCs were stimulated with NDV in concentrations as indicated for 48 hours, supernatants were collected, and concentrations of MIP-1β and RANTES were measured by ELISA. NDV infection significantly increased the production of RANTES (▦) as compared with controls (□; ^*P < .05; ^**P < .001). BMMCs were generated from 3 individual mice. Production of chemokines was analyzed in 2 independent experiments; data from one experiment are shown. Stimulation of PMCs with NDV (2.4 HA/mL) for 16 hours led to up-regulation of CD28 (F) and CD80 expression (G). Expression levels were analyzed on gated c-Kit⁺, S/T2⁺ double-positive cells. Gray shaded histograms show staining with isotype-matched control antibodies. Results are representative of 2 independent experiments. PMCs isolated from 3 to 4 individual mice were analyzed in each experiment.