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. 2004 Jan 29;23(3):552–563. doi: 10.1038/sj.emboj.7600067

Figure 3.

Figure 3

Endogenous TGF-β signaling is upregulated during the maturation phase. (A–E) mRNA expression patterns of ligands and receptors for TGF-β were determined during myoblastic or osteoblastic differentiation of C2C12 cells. C2C12 cells were treated as in Figure 2 and real-time RT–PCR analyses were performed. TGF-β1 gene (Tgfb1) (A), TGF-β2 gene (Tgfb2) (B), TGF-β3 gene (Tgfb3) (C), ALK5 gene (Tgfbr1) (D), and TGF-β type II receptor gene (Tgfbr2) (E) were determined. N, none; D, DMSO; SB, SB431542; B4, BMP-4. (F) C2C12 cells were pretreated or not with DMSO (0.01%) or SB431542 (1 μM) for 30 min in 5% FBS before stimulation. Cells were treated with BMP-4 (50 ng/ml) in combinations with or without DMSO or SB431542. TGF-β3 (1 ng/ml) was used as a control for phosphorylation of Smad2 and inhibition by SB431542. Whole-cell extracts were prepared at indicated time points, followed by immunoblotting. Activated Smad2 was detected by anti-phospho-Smad2 antibody. Smad2 and Smad3 were demonstrated by anti-Smad2/3 antibody. (G) Effects of BMP-4 and SB431542 were examined on different periods of osteoblastic differentiation of C2C12 cells. SB431542 (1 μM) was added to BMP-4 (50 ng/ml)-induced C2C12 cells at different periods as shown in the left panel. The middle panel demonstrates ALP staining. The right panel shows ALP activity.