IFN-γ activates the JAK-STAT pathway, which contributes to hepcidin induction. NHBE cells, grown at ALI, were exposed to IFN-γ (100 ng/ml) for 15, 60, and 120 minutes. Whole cell protein lysates (40 μg) were separated by SDS-polyacrylamide gel electrophoresis (7.5%) followed by immunoblotting using a phos-JAK2 antibody (Cell Signaling, Danvers, MA; 1:2000) then stripped and re-probed using a JAK2 antibody (Cell Signaling, 1:1000) to assess total JAK2 expression (A). Nuclear protein extracts (5 μg) from additional NHBE cells exposed to IFN-γ for 15 minutes were probed with a phos-STAT1 antibody and standardized to α-tubulin (B). NHBE cells pre-treated with a JAK2 inhibitor (AG490, 10 μM) prior to exposure to IFN-γ were harvested 24 hrs following exposure. RNA was isolated, reversed transcribed, and quantitative PCR performed. Relative abundance of hepcidin was normalized to 18s and expressed as fold change over induction ± SE and represent n = 4. * P < 0.001 relative to HBSS control cells, #P < 0.05 relative to IFN-γ stimulated cells.