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Figure 3


Fig. 3. Twist-1 inhibits BMP-induced Smad transcriptional activity with E-protein. (A) P19 cells were transiently transfected with 3GC2-Lux luciferase construct in combination with 50 ng of BMPR-IB(QD), Smad1, Smad4, E47 and increasing doses (12.5 or 50 ng) of Twist-1 expression construct. Cells were lysed and luciferase activity was assayed 24 hours after transfection (*P<0.01). The mean value of firefly luciferase and Renilla luciferase activity in cells transfected with luciferase expression plasmids was approximately 109,000 and 681,000 RLU, respectively. (B) P19 cells were transiently transfected with 3GC2-Lux luciferase construct in combination with 50 ng of BMPR-IB(QD), Smad1, Smad4, E47, Twist-1 and Id1 construct. Cells were lysed and luciferase activity was assayed 24 hours after transfection (*P<0.01). The mean value of firefly luciferase and Renilla luciferase activity in cells transfected with luciferase expression plasmids was approximately 51,000 and 199,000 RLU, respectively. (C) Flag-tagged full-length (FL) Twist-1 or deletion Twist-1 mutant (Twist-NBCT) and Myc-tagged E47 constructs were transfected into COS-7 cells. Lysates were immunoprecipitated using anti-Flag antibody and blotted with anti-Myc antibody. (D) P19 cells were transiently transfected with 3GC2-Lux luciferase construct in combination with 50 ng of BMPR-IB(QD), Smad1 and Smad4, E47, full-length Twist-1 and deletion Twist-1 mutant (Twist-NBCT) expression construct. Cells were lysed and luciferase activity was assayed 24 hours after transfection (**P<0.01, *P<0.05).





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