Iblot 7 min blotting system

Five-day differentiated 3D HIEs were solubilized in radioimmunoprecipitation assay buffer (Thermo Fisher Scientific) containing protease inhibitors (complete, mini, EDTA-free protease inhibitor cocktail tablets) and benzonase per the manufacturer’s instructions. Cells were lysed on ice for 20 mins and pelleted by centrifugation at 10,000 rpm (13,800g) in a Beckman Coulter J2-HS centrifuge. The cell lysates were frozen at −20°C until used. Cell lysates were prepared with Laemmli sample buffer containing β-mercaptoethanol for western blot analysis. Samples were heated for 10 mins at 70°C and loaded onto 4–20% polyacrylamide gradient gels (Bio-Rad) for electrophoretic separation in Tris-glycine-SDS buffer (Bio-Rad). Following electrophoresis, the proteins were transferred to nitrocellulose membranes using the iBlot 7-min blotting system (Thermo Fisher Scientific). Membranes were blocked for 1 h at room temperature in 1 × casein blocking buffer (Sigma) and immunoblotted using primary antibody to Claudin-2 (1:500 dilution, Thermo Fisher Scientific) followed by fluorescent secondary anti-mouse antibodies (1:10,000 dilution, LI-COR) in 1 × casein blocking buffer. Blots were washed three times with PBS– 0.5% Tween 20 and once with PBS prior to visualization using an Odyssey infrared imaging system (LI-COR).