HGF inhibits Smad signaling. (A) Detection of phosphorylated (P)–Smad 1, 5, and 8 in C2C12 cells treated for 45 minutes with BMP-2 (300 ng/mL) and/or HGF (100 ng/mL). (i) Control cells (P–Smad 1, 5, or 8 detected in 19% of the nuclei); (ii) BMP-2–treated cells (P–Smad 1, 5, or 8 detected in 57% of the nuclei); (iii) HGF-treated cells (P–Smad 1, 5, or 8 detected in 33 % of the nuclei); and (iv) BMP-2– and HGF-treated cells (P–Smad 1, 5, or 8 detected in 21% of the nuclei). (B) Detection of P–Smad 1, 5, and 8 by Western blotting of nuclear extracts of C2C12 cells treated for 45 minutes as indicated. (C) Luciferase activity of hMSCs transfected with BRE-luc stimulated for 5 hours as indicated. The concentrations of cytokines in (A-C) were 300 ng/mL BMP-2 and 100 ng/mL HGF.