IFN-β–induced STAT1 activation is responsible for the increased IP-10 production in diffDCs. (A) imDCs and diffDCs were stimulated with or without 500 ng/mL LPS for the indicated time. Total cell lysates were then electrophoresed and probed with phospho-STAT1 and STAT1, respectively. Actin expression was used as control to confirm that equal amounts of protein lysates were in each lane. (B) imDCs and diffDCs were pretreated with 50 μM fludarabine (STAT1 inhibitor) for 30 minutes, then stimulated with 500 ng/mL LPS for 24 hours. An equal amount of DMSO contained in medium was used as a negative control. The levels of IP-10 in the supernatants were determined by ELISA. *P < .05. (C) Effect of neutralizing antibody against mouse IFN-β on the phosphorylation of STAT1 in imDCs and diffDCs stimulated with or without LPS. imDCs and diffDCs were pretreated with 12.5 μg/mL neutralizing antibody of IFN-β for 30 minutes, then stimulated with 500 ng/mL LPS for 2 hours. The activation of STAT1 was analyzed by Western blotting. imDCs and diffDCs in medium alone and treated with IFN-β antibody were used as controls. (D-E) DCs were stimulated with various doses of recombinant mIFN-β or IFN-α for 24 hours. Then the level of IP-10 in the supernatants was determined by ELISA. Data are shown as means ± SD of 3 independent experiments. Each experiment was preformed at least 3 times. (F) imDCs and diffDCs were stimulated for 2 hours with or without 100 ng/mL LPS, CpG ODN, LTA, poly(I:C), or HSP70. Phosphorylation of STAT1 in cell lysates was analyzed by Western blotting. Actin expression was used as control to confirm that equal amounts of protein lysates were in each lane.