(A) FLAG-tagged Smad1 or Smad8 were transiently expressed in C3H10T1/2, and BMP2, TGFβ1, or GDF5 were added for 30 minutes at the indicated concentrations. After Western blotting, ligand-dependent phosphorylation of Smads was shown by antibodies specific for pSmad1, -5, and -8. The expression rate of the Smads was determined by Western blotting using anti-FLAG antibodies. (B) Evaluation of the Smad1 and Smad8 transactivation potential in HEK 293T cells, in which a GAL4 reporter with the GAL4 DNA-binding domain was fused to Smad1WT and Smad8WT proteins as described in Figure 3B. Pooled data from 3 independent experiments are presented. BMP2 led to efficient activation of the GAL4-Smad1 fusion protein, GDF5 was less effective, and TGFβ1 did not exhibit notable activation. The GAL4-Smad8 fusion protein was activated, albeit to a markedly lower extent, by BMP2, but not by TGFβ1 or GDF5. The expression level of the GAL4-Smad fusions was comparable as assessed by Western analyses of cellular extracts and blotting with anti-GAL4 antibodies. (C) Compared with Smad1, Smad8 exhibited a lower BMP2-dependent activation potential in C3H10T1/2. FLAG-tagged Smad1 or Smad8 were transiently expressed in C3H10T1/2, and BMP2 (200 ng/ml) was added for 30 minutes. Top: After Western blotting, BMP2-dependent phosphorylation of Smads is shown by anti-pSmad1, -5, and -8 antibodies. Bottom: Expression rates of the Smads were determined by Western blotting using anti-FLAG antibodies. In the graph at right, the level of BMP2-dependent Smad activation in C3H10T1/2 was evaluated by densitometric scanning.