Epithelial Barrier Function Regulation

To evaluate the mechanism of OSM and IL-6 on epithelial barrier function, we evaluated in vitro the expression level of occludin and ZO-1 at protein levels in cytokine-stimulated HNECs. 1 ng/mL, 10 ng/mL, and 100 ng/mL were the concentrations chosen according to previous studies that showed an IL effect on epithelial cells [28 (link),37 (link),60 (link),73 (link)] to identify a dose–response effect. After 48 h of stimulation, Western blot was performed as previously described. Immunodetection of occludin, ZO-1, and GAPDH was performed by co-incubation with a rabbit anti-occludin antibody (Ab) (Invitrogen, MA, USA), a mouse anti-ZO-1 Ab (Invitrogen, Waltham, MA, USA), and mouse anti-GAPDH mAb (Novus, clone 2D4A7), followed by co-incubation with an anti-rabbit and an anti-mouse IgG peroxidase-conjugated polyclonal Abs (Sigma-Aldrich, St. Louis, MO, USA).