Figure 6.

Monovalent FSTL3-Fc protein has longer serum half-life and increases muscle mass after systemic administration in mice

(A) Schematic presentation of monovalent FSTL3-Fc protein used in this study. See also Supplemental information for cDNA sequence.

(B) Ligand neutralization by mono-FSTL3-Fc, measured in Hs578T-reporter cells with 9×CAGA-Luc for SMAD2/3. The data represent mean ± SD from n = 3 independent experiments.

(C) Ligand neutralization by mono-FSTL3-Fc, measured in HMEC-1-reporter cells with BRE-Luc for SMAD1/5/8. The data represent mean ± SD from n = 3 independent experiments.

(D) Ligand-binding parameters for mono-FSTL3-Fc as determined by reporter cell-based assay. ∗ indicates data from Figure 2C, ∗∗ indicates data from Figure S8A, and ∗∗∗ indicates data from Figure 2D.

(E) Immunoblot analysis for FSTL3 in reduced serum taken from mice at indicated time points after i.p. or i.v. injection with mono-FSTL3-Fc (10 mg/kg) (n = 2 independent experiments. #, ψ, Δ, or ▲ represents each mouse).

(F) Time course of serum concentration of FSTL3-Fc or control Fc measured by anti-human IgG Fc ELISA. Serum was taken at the indicated time points after injection with monovalent- or bivalent-FSTL3-Fc or control Fc (10 mg/kg). The data represent mean ± SD from n = 3 independent experiments, except for bi-FSTL3-Fc, at 96 hr (n = 2, because of anesthesia-related death). Differences between the conditions were analyzed using analysis of variance (ANOVA) followed by Tukey-Kramer post hoc test for multiple comparison; n.s., not significant, ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001.