FIGURE 9.

fad104 inhibits the translocation of Smad1 to the nucleus. A, the Smad1/5/8 phosphorylation levels after BMP2 treatment in HeLa cells infected with FAD104 or LacZ. Nearly confluent cells were infected with each adenovirus. After 24 h, the cells were starved for 6 h and then treated with 100 ng/ml BMP2. At 30 and 120 min after BMP2 treatment, the phosphorylation and protein levels of Smad1/5/8 were determined by Western blotting. β-Actin expression was used as a control. The figures show typical results, and similar results were obtained in at least two independent experiments. B, FLAG-tagged Smad1 expression plasmid was transiently introduced into HeLa cells with Myc-tagged FAD104 expression plasmid or empty vector. After transfection, the cells were starved for 6 h and treated BMP2 for 2 h. After conducting immunofluorescent staining, the signals of FLAG-Smad1 (red), Myc-FAD104 (green), and DAPI (blue) were detected with fluorescence microscopy. C, FLAG-tagged Smad1 expression plasmid and Myc-tagged FAD104 expression plasmid were transiently introduced into HeLa cells. After transfection, the cells were starved for 6 h and treated with BMP2 for 2 h. The signals of FLAG-Smad1 (red), Myc-FAD104 (green), and DAPI (blue) were detected with fluorescence microscopy.