Fig. 7

Binding of GDF11 to the type I receptor ALK5. a Basic Native-PAGE (12%) of GDF8 (top) and GDF11 (bottom) ligand, receptor, and ligand:receptor complexes. To test for ligand-receptor complex formation, receptors were first diluted into 20 mM HEPES pH 7.4 followed by addition of the ligand. The ligand:receptor mixtures were held at room temperature (25 °C) for 15 min before being loaded and run in the gel. Amount loaded for each protein: ligand (high conc.; lane 1), ALK5-ECD (lane 2), ligand + ALK5-ECD (lane 3), ActRIIB-ECD (lane 5) = 3 μg each; ligand (low conc.; lane 4) = 1.5 μg; ligand + ActRIIB-ECD (lane 6) = 1.5 μg ligand and 3 μg ActRIIB-ECD; for ALK5-ECD titration (blue triangle; lanes 7–10) ligand = 1.5 μg, ActRIIB-ECD = 3 μg, and 1, 2, 4, or 8 μg ALK5-ECD. Proteins were visualized by colloidal Coomassie stain. b Surface plasmon resonance (SPR) sensorgrams of GDF8 (left) and GDF11 (right) binding to Fc-ALK5-ECD. Fc-ALK5-ECD was captured by Protein A that was amine coupled to a CM5 chip. Serial dilutions (500–15.265 nM) of each ligand alone (top) or in the presence (bottom) of a twofold molar excess of ActRIIB-ECD were allowed to associate for 120 s at a 70 μL/min flow rate followed by a 280 s dissociation at 37 °C. Sensorgrams were double referenced using an average of two 0 nM ligand injections. Data information: Ligand sources: GDF8 and GDF11, gift from Acceleron Pharma; Activin A, Activin B, and TGFβ3, produced and purified as described in “Methods”