Fig. 4.

Characterization of bacterially produced GDF8 prodomains and purified prodomain:ligand complexes. (A) Representative IC₅₀ curve of serially diluted bacterially expressed prodomains mixed with exogenous, mammalian-derived GDF8^apo (0.62 nM) and added to HEK293 (CAGA)₁₂ cells. Fraction activation was calculated using the signal of GDF8^apo treated with the prodomain divided by GDF8^apo alone or maximum signal. Data were fit to a nonlinear regression with variable slope to determine the IC₅₀. (B) EC₅₀ curves of reformed GDF8 (mammalian-derived) prodomain (bacterial derived) complexes denoted with Rbac superscript. Data were fit by nonlinear regression to a variable slope to determine the EC₅₀. (C) Representative derivative plot of melt curves from 24 to 100 °C generated by thermal shift and reported as fluorescent units. (D) Representative T_m (C^o) for each mutant shown in C. All experiments were performed at least twice where individual points were measured in triplicate for A and B and duplicate for C and D. All data are shown as mean ± SEM.