Mouse anti gapdh mab

After 48 h of stimulation, fibroblast lysis was performed as previously described^{61 (link)}. After separation on a 12% SDS-PAGE gel, proteins were transferred to nitrocellulose membranes (GE Healthcare, Chicago, IL, USA) by electroblotting. Immunodetection of αSMA and GAPDH was performed by co-incubation with rabbit anti-αSMA Ab (Novus Biologicals, Centennial, CO, USA) and mouse anti-GAPDH mAb (Novus, clone 2D4A7), followed by co-incubation with anti-rabbit and anti-mouse IgG peroxidase-conjugated polyclonal Abs (Sigma-Aldrich, St. Louis, MO, USA). Peroxidase activity was detected by chemiluminescence (Luminata HRP substrate from Merck Millipore Burlington, MA, USA) and analysed using an LAS-3000 imaging system (Fujifilm, Tokyo, Japan) followed by quantification using Multi Gauge V3.0 software (Fujifilm). Each membrane was exposed for 10 sec and 30 sec for GAPDH and αSMA quantification, respectively. The ratios of αSMA/GAPDH were calculated and are shown in the corresponding figures. The experiment was repeated four times, and the images are representative of one experiment.

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).