Bca protein assay method

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).

For Western blot analyses, subcellular fractions were incubated with RIPA buffer (10 mM Tris–Cl (pH 8.0), 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, R0278, Sigma) in the presence of protease inhibitors (Halt™ Protease and Phosphatase Inhibitor Cocktail (100×), Promega) for 20 min on ice, followed by centrifugation at 10,000×g for 10 min at 4 °C. Supernatant was saved and the protein concentrations determined using the BCA protein assay method (Bio-Rad). For the immunoprecipitation (IP) experiments a milder IP lysis buffer (25 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100) was used.