International Journal of Stem Cells : eISSN 2005-5447

Fig. 4.

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Fig. 4. TGF-β triggers Smad2 phosphorylation and transactivation. (A) 3TP luciferase reporter activity in SM10s cells treated with vehicle control or TGF-β2. Cells were transiently transfected with 3TP-lux and pRLSV40 using Metafectene. Twenty-four hours post-transfection, cells were treated with vehicle or TGF-β2 (5 ng/ml) for 72 hours. Luciferase activity was analyzed using the dual luciferase assay system. The 3TP-lux transactivation values were normalized to the constitutively active reporter (pRLSV40). (B) Western blotting analysis of Smad2, Smad3, and Smad4 in TGF-β2 or vehicle-treated SM10 cells. COS-7 cells were used as a positive control. (C) Western blotting analysis of Phospho-Smad3 in TGF-β2 or vehicle-treated SM10 cells. COS-7 cells transiently transfected with the plasmid, pXIX-Smad3 and treated with TGF-β were used as a positive control. (D) Western blotting analysis of Phospho-Smad2 in the TGF-β2, Activin A, or vehicle-treated SM10 cells. (E) ARE luciferase reporter activity in SM10 cells transiently transfected with pCDNA3 control or pRK5-Smad2 and treated with vehicle control or TGF-β2. Cells were transiently transfected with ARE-lux, pLv-CMV-hFAST-1, and pRLSV40 using Metafectene. Twenty-four hours post-transfection, cells were treated with vehicle or TGF-β2 (5 ng/ml) for 72 hours. Luciferase activity was analyzed using the dual luciferase assay system. The ARE-lux transactivation values were normalized to the constitutively active reporter (pRLSV40). (F) ARE luciferase reporter activity in SM10 cells transiently transfected with pCDNA3 control or pRK5-Smad7 and treated with vehicle control or TGF-β2. Cells were transiently transfected with ARE-lux, pLv-CMV-hFAST-1, and pRLSV40 using Metafectene. Twenty-four hours post-transfection, cells were treated with vehicle or TGF-β2 (5 ng/ml) for 72 hours. Luciferase activity was analyzed using the dual luciferase assay system. The ARE-lux transactivation values were normalized to the constitutively active reporter (pRLSV40). Error bars represent standard error of the mean. *p<0.05.
International Journal of Stem Cells 2018;11:111-20 https://doi.org/10.15283/ijsc17069
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