Total proteins were extracted from transfected HRCECs and quantitated using the bicinchoninic acid (BCA) method. An amount of 50 μg total protein was mixed with loading buffer, denatured for 5 minutes at 60°C, cooled, centrifuged for 5 minutes, and separated by sodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Antibodies of mouse anti-human ALK1 monoclonal antibody (1500× dilution; DaAn Gene Co., Guangzhou, China), anti-human ANG2 (1500× dilution; BD BioSciences, San Jose, CA), anti-human occludin (1000× dilution; BD BioSciences), and anti-human ALK5 monoclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) were used for probing the proteins. A secondary antibody (1000× dilution) was then applied, and the signal was revealed by chemiluminescence. The same polyvinylidene difluoride (PVDF) membrane was reused to detect β-actin by incubation with mouse anti-human actin antibody (Gene Co., Hong Kong, China) which was used as an internal control. The bands observed on the films were analyzed by automatic image analysis, and the integrated optical density (OPTDI) of each ALK1 band was normalized to the OPTDI value of the corresponding β-actin band from the same sample.