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. 2003 Oct;23(20):7230–7242. doi: 10.1128/MCB.23.20.7230-7242.2003

FIG. 2.

FIG. 2.

Myostatin signals through a TGF-β/activin signaling pathway. (A to C) Myostatin (10 nM) activates the TGF-β-responsive reporters 3TP-Lux, A3-Lux, and SBE4-Lux but not the BMP-responsive reporter I-BRE-Lux. HepG2 (A and C), C3H 10T1/2 (B and C), and 3T3-L1 (B) cells were transiently transfected with the indicated reporter constructs, and luciferase activity in cells treated with TGF-β, myostatin, or BMP7 or left untreated was determined. (D to F). Myostatin (10 nM) induces phosphorylation of endogenous Smad2 and Smad3. 3T3-L1 cells were incubated with TGF-β, myostatin, or BMP7 for 30 min, and cell lysates were subjected to anti-Smad2/Smad3 (D and E) or anti-Smad1 (F) immunoprecipitation followed by immunoblotting with anti-phosphospecific Smad2 (PSmad2), anti-phosphospecific Smad2/Smad3 (PSmad2/3), or anti-phosphospecific Smad1 (PSmad 1) antibodies. Total Smad proteins were detected by anti-Smad2 (D and E), anti-Smad3 (E), and anti-Smad1 (F) antibodies (lower panels).