STAT3 activation is necessary for AC-induced inhibition of NF-κB activation in DCs. MerTK^+/+ BMDCs (A) or sDCs (B) were incubated with the STAT3 inhibitors Jsi124 (10 nM) or sttatic (20 nM) for 1 hour, cocultured with ACs or left untreated for 3 hours, and stimulated with LPS (50 ng/mL) for 0.5 hours. Nuclear NF-κB and SP-1 DNA-binding activity were determined via EMSA, and IκBα and β actin protein expression was detected by Western blot. (C-D) MerTK^+/+ BMDCs were transfected with STAT3-specific or scrambled control siRNA, cocultured with ACs for 3 hours, stimulated with LPS, and either (C) nuclear NF-κB and SP-1 DNA-binding activity and IκBα or β actin protein was measured as described for panels A and B or (D) IL-12 secretion was measured by enzyme-linked immunosorbent assay; *P < 10⁻³, DCs treated with STAT3-specific siRNA versus mock-transfected DCs or DCs transfected with control siRNA (Student t test). Error bars indicate ± SEM. (E) MerTK^+/+ BMDCs were pretreated with the PI3K inhibitors Wort and LY for 1 hour and incubated with ACs for the indicated times, and pSTAT3 and STAT3 protein was measured. (F) MerTK^+/+ or MerTK^−/− BMDCs were incubated with ACs for the indicated times, and p-STAT1 and STAT1 proteins were measured by Western blot. (G) MerTK^+/+ BMDCs were treated with STAT1 inhibitor fludarabine (20 μM) for 1 hour, and cocultured with ACs for 3 hours or left untreated, and stimulated with LPS (50 ng/mL). Nuclear NF-κB or SP-1 DNA-binding activity, and IκBα and β actin protein were measured as described for panels A-B. Data are representative of a minimum of 3 experiments.