Anti gpc3

Western blotting was performed for the analysis of expression of GPC3 in HepG2 and HLE cells. Briefly, cells were lysed in RIPA lysis buffer (Beyotime Institute of Biotechnology, Beijing, China) and 40 µg protein was utilized for each western blot. BCA assay was used to determinate the protein concentrations (Beyotime Institute of Biotechnology). Electrophoretic transfer of proteins was performed from 10% SDS-PAGE gels onto nitrocellulose membranes. Membranes were blocked by immersion in 5% non-fat milk (w/v)/PBS for 1 h at room temperature, and then incubated at 4°C overnight with anti-GPC3 (dilution, 1:500; catalog no., sc-390587) and β-actin monoclonal antibodies (dilution, 1:1,000; catalog no., sc-130656; both Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Subsequent to rinsing with PBST for three times, membranes were incubated at 37°C with a horseradish peroxidase-conjugated IgG secondary antibody for 1 h. Immunocomplexes were visualized by incubation of the membranes with the Enhanced Chemiluminescence kit (catalog no., 32109, Thermo Fisher Scientific, Inc.) at room temperature for 1 min according to the manufacturer's protocol.

The expression level of GPC-3 was determined in the liver tissue and HCC cell lysates using western blotting. The lysate samples (10 µg) were subjected to SDS-PAGE following the methods of Laemmli (36 (link)). Next, the resolved proteins were subjected to western blotting, following the methods of Towbin et al (37 (link)). The membrane was incubated with the following primary antibodies: Anti-GPC-3 (1:500) and anti-neomycin phosphotransferase II (1:500) (both Santa Cruz Biotechnology, Inc.). β-actin was used as the loading control in western blot analysis. The immunoreactive bands were visualized using an enhanced chemiluminescence detection system (Funakoshi Co., Ltd.) on an X-ray film.