FIGURE 5.

Ectopic expression of SCP4 attenuates BMP signaling. A, SCP4 is localized in the nucleus. FLAG-PPM1A or FLAG-SCP4 was transfected into HeLa cells, and expressed proteins were detected by immunofluorescence with anti-FLAG antibodies. DNA was stained using DAPI dye. B, SCP4 reduces the Smad1-Smad4 complex formation. Myc-Smad1 and HA-Smad4 were co-transfected with WT SCP4 or SCP4 mutant SCP4DN in HEK293T cells. Smad1 was immunoprecipitated (IP) by anti-Myc antibodies, and the Smad1-bound Smad4 was detected by Western blotting (IB) with anti-HA antibodies. WCL, whole cell lysate. C, SCP4 inhibits BMP2-induced Id1-luc reporter activity. C2C12 cells were transfected with a phosphatase, together with Id1-luc reporter plasmid. BMP2 treatment and luciferase assays were done as described under “Experimental Procedures.” D, SCP4 inhibits BMP2-induced Id1-luc reporter expression in ATDC5 cells. Experiment was carried out as described for panel C. E, SCP4 inhibits BMP2-induced Id1-luc reporter expression in C3H10T1/2 cells. Experiment was carried out as described for panel C. F, SCP4 has no effect on TGF-β-induced synthetic SBE-luc reporter expression. HaCaT cells were transfected with SCP4, together with SBE-luc reporter plasmid, and then treated with 2 ng/ml TGF-β for 8 h. Experiment was carried out as described for panel C. Cntl, control. G, SCP4 has no effect on TGF-β-induced natural PAI-1-luc reporter expression in HaCaT cells. Experiment was carried out as described for panel F. Error bars in panels C–G indicate means ± S.E.