Fig 9.

Latent myostatin binds WFIKKN1 but not WFIKKN2. In Ni²⁺-affinity pull-down assays, 1 μm latent myostatin was incubated for 1 h with 2 μm His-tagged WFIKKN1 or 2 μm His-tagged WFIKKN2 in NaCl/P_i containing 50 mm imidazole, 0.1% Tween-20 and 100 μm phenylmethanesulfonyl fluoride (pH 7.5), and the solutions were then mixed with 20 μL of Ni²⁺–nitrilotriacetic acid resin. After 15 min of agitation, the resin was washed with NaCl/P_i, 50 mm imidazole, 0.5% Tween-20 and 100 μm phenylmethanesulfonyl fluoride (pH 7.5), and the bound proteins were eluted with NaCl/P_i and 500 mm imidazole (pH 7.5). The eluted samples were analyzed by SDS/PAGE, and the proteins were visualized by staining with Coomassie Brilliant Blue and by western blotting with specific antibodies against myostatin prodomain (anti-prodomain) and against mature myostatin (anti-myostatin). LM, latent myostatin. The numbers indicate the molecular mass values of proteins of the Low Molecular Weight Calibration Kit. In the upper panel, the proteins were visualized by staining with Coomassie Brilliant Blue; in the lower panels, the proteins were visualized by western blotting.