IF staining and Western blot for MUC5AC/β-Tubulin IV in the NHBE. IL-13 mediated a concentration dependent increase in MUC5AC protein levels in the NHBE as seen by (A) Immunofluorescence, where at day 22 and day 28, 1 and 7 days after withdrawal of 14 day treatment with IL-13 (1,10 ng/ml for day 22 and 10 ng/ml for day 28) the staining for MUC5AC is higher compared to the untreated NHBE (0 ng/ml IL-13) (n = 3 donors of NHBE; grown in duplicate; with 3–6 wells per condition; scale bar = 20 μm). DAPI staining of the nuclei showed similar number of cells in all conditions (data not shown). (B) Levels of MUC5AC protein show a dose dependent increase via western blot at day 22 and day 28. Also during co-culture with the NHLF the dose dependent increase of MUC5AC is maintained at day 25 and not at day 31. Levels of β-Tubulin IV protein in the NHBE shown an inverse dependence on IL-13 concentration at days 22 and day 28 with levels remaining constant at day 25 and day 31 of co-culture with NHLF. Images are representative from 3 NHBE donors. (C, D) Quantification of MUC5AC/β-Actin and β-Tubulin IV/β-Actin levels relative to IL-13 concentration of 0 ng/ml at day 22 condition, show a dose dependent increase with IL-13 concentration at day 22,28 and 25 for MUC5AC and dose dependent decrease at day 22, 28 and 31 for β-Tubulin IV (Statistical difference between conditions by ANOVA # p < 0.01).