Bafilomycin A1 inhibits both R-848– and influenza-mediated signaling. (A) Human neutrophils (10⁶ cells) were incubated with GM-CSF (100 U/mL) for 30 minutes, with the indicated concentrations of bafilomycin A1 for 30 minutes, and then stimulated with influenza virus (X31, 100 000 HA). Supernatants were collected at 24 hours and cytokines measured by ELISA. (B) HEK/TLR8 cells (5 × 10⁴ cells) were transfected with reporter plasmids, incubated with the indicated concentration of bafilomycin A1 for 30 minutes, and then stimulated with either R-848 (10 μM) or IL-1β (100 ng/mL). Cells were lysed 8 hours later. The readout is the fold increase of firefly luciferase/Renilla luciferase ratio over the unstimulated control. Error bars represent SD of triplicate samples.