2.10. Protein Detection, Electrophoresis, and Western Blot Analysis
SECONDARY, ALDRICH
The cells were scraped using ice-cold RIPA buffer in the presence of a protease and phosphatase inhibitor cocktail (10 μg/mL; Thermo Fisher Scientific, Waltham, MA, USA) and sonicated three times. Following incubation on ice for 30 min, the samples were centrifuged at 10,000× The Bradford colorimetric assay was used to determine the protein concentration and equal amounts of cell proteins (30 μg/lane) were separated by SDS-PAGE (10–12% polyacrylamide gel) and then electro-transferred onto nitrocellulose membranes by a Trans-Blot Turbo (Mini Nitrocellulose, Bio-Rad, Hercules, CA, USA). Incubation was performed overnight at 4 °C with primary antibodies: rabbit polyclonal anti-occludin (Sigma Aldrich) and mouse monoclonal anti-actin (Santa-Cruz Biotechnologies, Dallas, TX, USA). HRP-conjugated secondary antibodies (1:3000; Bio-Rad, CA, USA) were incubated with the membranes for 1 h. The blots were then detected using an enhanced chemiluminescence substrate (ECL Substrates, Bio-Rad, CA, USA). Densitometric analysis was performed using Image Lab software 6.1.0. The results were expressed as arbitrary units (A.U.) and represented as the mean of three independent experiments.