Figure 3.

Overexpression of Smad6 inhibited activation of Smad1 (A), BMP2-regulated induction of PPARγ (B), and adipogenesis (C and D). (A) C3H10T1/2 cells infected with control or Flag-Smad6 adenovirus at 100 multiplicity of infection were starved with 0.2% fetal calf serum-DMEM for 16 h and then stimulated with or without BMP2 (300 ng/ml) for 10 min and lysed. The cell lysates were immunprecipitated with anti-Smad1 antibody and immunoblotted with anti-phsophoserine antibody (top). The amount of Smad1 in the immunoprecipitates was determined by immunoblotting with anti-Smad1 (second panel). The lysates were also determined by immunoblotting with antiphsopho-p38 (third panel) or antip38 (forth panel) antibodies. The expression of Flag-Smad6 was determined by immunoblotting with anti-Flag antibody (bottom). (B) C3H10T1/2 cells infected with control or Flag-Smad6 adenovirus at 100 multiplicity of infection were incubated with or without BMP2 (300 ng/ml) for 4 d and lysed. The lysates were determined by immunoblotting with anti-PPARγ mAb followed by immunoprecipitation with anti-PPARγ polyclonal antibody (top). The lysates were also determined by immunoblotting with anti-Flag (middle) or anti-β-actin antibodies (bottom). (C and D) 10T1/2 cells infected with control or Flag-Smad6 adenovirus at 100 multiplicity of infection were incubated with or without BMP2 (300 ng/ml) for 10 d. The cells were stained with Oil Red O, photographed under a microscope, and the areas stained with Oil Red O in the cells were assessed.