TFPI binds to GPC3 expressed on the cell surface of murine as well as human HSPCs. (A) Co-IP experiments were performed for TFPI (top panel) or GPC3 (bottom panel) using mouse BM-derived lin⁻ cells incubated with or without TFPI for 3 hours. Western blotting was performed to detect GPC3 or TFPI in the immunoprecipitated protein, respectively. (B) Lin⁻ BM cells from WT or GPC3^−/− mice were used for flow cytometric detection of TFPI bound to HSPCs. The cells were incubated with or without TFPI for 3 hours and stained with specific anti-TFPI antibodies. TFPI binding to the HSPCs was detected by performing flow cytometry. (C) WT and GPC3^−/− mouse BM-derived KLS cells were incubated for 3 hours with or without TFPI. Immunostaining was performed to detect binding of TFPI. All results are representative of at least 3 experiments.