Isolation of Native MUC5AC and MUC5B Mucins.

MUC5AC and MUC5B monomucin Calu3 cell cultures were washed once per week by apical administration of 100 µL phosphate-buffered saline (PBS), incubation for 30 min, and then collection of the PBS along with harvested secretions. Culture secretions were pooled together and subjected to isopycnic density gradient centrifugation (initial density ρ = 1.35 g/mL) for mucin isolation (5, 34). Fractions were probed with MUC5AC and MUC5B antibodies (34). Mucin-containing fractions (around the density 1.35 to 1.40 g/mL) were pooled for further analyses.