Anti gpc3 antibody

DSF-treated HCC cells were subjected to Western blot analysis using anti-p38 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-p38 (Cell Signaling Technology), and anti-tubulin (Oncogene Science, Cambridge, MA) antibodies. ALDH2-knockdown cells and ALDH1-and ALDH2-double knockdown cells were subjected to Western blotting using anti-ALDH1 (BD Biosciences) and anti-ALDH2 (Abcam, Cambridge, MA) antibodies. GPC3-knockdown cells selected by cell sorting for enhanced green fluorescent protein (EGFP) expression were also subjected to Western blot analysis using anti-GPC3 antibody (Santa Cruz Biotechnology).

Cells were seeded in 6-well plates (3 × 10⁵ cells/well) the day before use. Cells were then transfected with plasmid (2.5 μg/well) using Lipofectamine 2000 (Invitrogen). At 24 h post-transfection, cell lysates were prepared using an IP kit (Thermo Cat# 26148), according to the manufacturer’s instructions. Briefly, after lysis with ice-cold IP Lysis buffer containing a protease inhibitor cocktail, 500 μg of cell lysate was subjected to IP with 2 μg of anti-GFP antibody (Abcam, cat# ab1218), anti-GPC3 antibody (Santa Cruz, cat# sc-65443), anti-CD44 (Abcam, cat# 6124), or anti-HA antibody (Millipore, cat# 05-904) coupled to a resin. If necessary, transfected HeLa cells were dissociated with non-enzymatic cell dissociation solution and treated with 500 mIU/ml heparinase III or 500 mIU/ml chondroitinase ABC for 2 h at 37 °C, followed by the treatment with 5 μM 3D8 scFv for 6 h at 37 °C. Then, cell lysates were prepared using the IP kit. Protein concentration in the cell lysates was measured using a BCA Protein assay kit (Thermo Cat# 23227). After washing the resin, the immunoprecipitated proteins were eluted and resolved on 4–20% gradient SDS-PAGE gels, followed by immunoblotting of proteoglycans and 3D8 scFv with antibodies specific for SDC2 (Invitrogen, cat# 36-6200), CD44 (Abcam, cat# 6124), GPC3, His tag (Abcam, cat# ab18184), or the HA tag.