Western blotting

To examine the expression levels of proteins in HLECs under the influence of fucoidan, cells were first treated with fucoidan for 24 h and then lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (Thermo scientific, USA). Equal amounts of protein (40 μg) were measured using the Bradford method and resolved by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electro-transferred onto nitrocellulose (NC) membranes. Membranes were incubated overnight at 4°C with relevant primary and peroxidase-conjugated secondary antibodies; finally, the bands were detected with an ECL system (Bio-Rad Laboratories, CA, USA).