Figure 3. Analysis of Tll1 astacin domain activity vs GDF8 prodomain states.

(A) Fluorogenic peptide incubated with the astacin domain and treated with a titration GDF8 prodomain, plotted as fold specific activity. (B) Latent GDF8 treated with a titration of the Tll1 astacin domain. Data were fit by non-linear regression with a variable slope to generate the EC₅₀ curve. (C) Luciferase assay of latent GDF8 (GDF8^L), acid-activated GDF8 procomplex (GDF8^AA), and a mixture of GDF8 prodomain:mature domain in a 3 : 1 molar ratio, respectively, treated with or without the Tll1 astacin domain. The DNA transfected (Trx) and exogenous (Exo) protein added (if applicable) are denoted in the top left of each luciferase assay. (D) Anti-GDF8 prodomain western blot of GDF8^L, GDF8^AA, or GDF8 prodomain alone (Pro^WT) treated with the Tll1 astacin domain for 30 min or 1 h. Experiments a–c were conducted at least three times with experimental triplicate.