Protein Extraction and Western Blot Analysis

As previously described^{24 (link)}, protein was isolated from differentiated AEC cultures in situ from the transwell membrane surface using M-Per mammalian cell protein lysis reagent and Halt® protease and phosphatase inhibitor cocktail (both Thermo Scientific, Victoria, Australia). Protein samples were quantified using the BCA protein assay method (Bio-Rad, Victoria, Australia), and 10 μg electrophoresed using Novex® 4–12% gradient Bis–Tris denaturing gels (Life Technologies, Victoria, Australia) and electroblotted to Trans-Blot® Turbo nitrocellulose membranes (Bio-Rad). Membranes were blocked in 5% diploma skim milk and probed overnight at 4 °C with primary antibodies directed to Claudin-1 (37–4900), Occludin-1 (71–1500), ZO-1 (33–9100) (all Thermo Fisher Scientific, Waltham, MA, USA), or β-actin (A1978; Sigma-Aldrich Co., St Louis, MO, USA). Secondary antibody incubation was 1 h at RT with horse radish peroxidase-labelled secondary antibody (R&D Systems, MN, USA). Chemiluminescent imaging was performed using the LAS-3000 platform and histogram densitometry was performed using Multi Gauge software (V3.1 Fujifilm, Tokyo, Japan).