Endoglin-GFP Influence on BMP9/10 Signaling

C2C12-BRE cells [51 (link)] were seeded at 30,000 cells/well in 96-well plates and grown for 3 days in DMEM containing 10% heat-inactivated FBS and antibiotic/antimycotic (Thermo Fisher Scientific, Waltham, MA, USA). Cells were then washed once with DMEM containing 0.1% FBS and antibiotic/antimycotic; after aspiration of the washed, they were incubated in fresh 0.1% FBS overnight. The endoglin–GFP protein was expressed using the EGFP–N1–EndoglinCDS1 plasmid and purified with Strep-Tactin^®XT (GenScript Biotech, Leiden, The Netherlands). As the provided dilution of the protein stock (30 µg/mL) was limiting, media for the incubations were prepared using 10X M199 (Sigma-Aldrich, St. Louis, MO, USA). Dilutions of BMP9 or BMP10 (both from R&D Systems, Minneapolis, MN, USA) were incubated in the absence or presence of endoglin–GFP in M199 containing 1.5 mg/mL sodium bicarbonate, 4 mM L-glutamine, 25 mM Hepes, and 0.5% recombinant human serum albumin (Sigma-Aldrich, St. Louis, MO, USA). ALK1-Fc (R&D Systems, Minneapolis, MN, USA) was included as a positive control.