Immunoblotting and Immunoprecipitation.

Cells were harvested, vortexed in ice-cold lysis buffer (Cell Signaling Technology), and clarified by centrifugation at 10,000 × g for 10 min at 4 °C. Protein concentration was measured using a Coomassie blue assay (Pierce) following the manufacturer’s specifications. A total of 20 μg of cell lysates was loaded onto a 4–20% SDS/PAGE gel for electrophoresis. The anti-GPC2 antibody for Western blotting was purchased from Santa Cruz Biotechnology. The anti-active β-catenin and N-Myc antibodies were obtained from Millipore. The anti-Wnt3a and Wnt11 antibodies were purchased from Abcam. All other antibodies, including cleaved PARP, total β-catenin, β-actin, and GAPDH were obtained from Cell Signaling Technology.

Fifty micrograms of GPC2–hFc protein was incubated with Wnt3a CM for 4 h on ice. Protein A-Agarose beads (Roche) were added to the immune complex and incubate overnight at 4 °C. Immune complexes were washed three times with lysis buffer and subjected to immunoblotting with anti-Wnt3a antibody.