Fig. 3.
Fig. 3. Selective inhibition by Smad7 of signaling of type I activin receptor. / Chinese hamster ovary (CHO) K1 cells (A) and F5-5 mouse erythroid leukemia cells (B) were transfected with 300 ng of reporter construct p3TP-Lux, 100 ng of the internal control pRL-1PGK, and 200 ng of either control vector pactEF or Smad7 expression vector pactEF-Smad7 and then cultured in the absence or presence of 1 nmol/L activin. Transcriptional activity of the 3TP promoter was measured as firefly luciferase activity in the cell lysate. Values were normalized by comparison with values for sea pansy luciferase activity, which was driven by the constitutive promoter of the mouse phosphoglycerate kinase gene in pRL-1PGK. Results are expressed as the increase in induction (x-fold) compared with the value in the untreated vector control. Values are the mean and SD from at least 3 assays. (C) ST2 mouse fibroblast cells rendered highly responsive to activin by transfection of activin receptors in the p3TP-Lux assays. In parent cells, activin treatment produced only a weak activation of the 3TP promoter. ST2 cells were transiently transfected with 300 ng of p3TP-Lux, 100 ng of pRL-1PGK, and 50 ng each of expression vector for ActR-II (pactEF-ActR-II) and either ActR-I (pactEF-ActR-I) or ActR-IB (pactEF-ActR-IB) and then cultured in the absence or presence of activin. Luciferase activity was determined as described above. (D) Effects of transfection of activin receptors into NMuMG mouse mammary epithelial cells. Cells were treated and transcriptional activation was assessed as described in (C). (E and F) Efficient suppression by Smad7 of p3TP-Lux activation by activin in the cells producing ActR-I but only inefficient suppression in the cells producing ActR-IB. Increasing amounts of Smad7 vector (0-200 ng) were cotransfected with vectors for ActR-II and either ActR-I or ActR-IB (50 ng each) into ST2 cells (E) and NMuMG cells (F). After treatment with activin, the cells were harvested for the luciferase assay described above. (G) Immunoblot showing comparable levels of the type I receptor proteins, ActR-I and ActR-IB, in transiently transfected cells. NMuMG cells were transfected with carboxy-terminal Flag-tagged ActR-I or ActR-IB in the absence or presence of Smad7. A fixed amount (20 μg each) of total protein from the whole-cell lysate was analyzed by gel electrophoresis and subsequent immunoblotting with anti-Flag monoclonal antibody.

Selective inhibition by Smad7 of signaling of type I activin receptor.

Chinese hamster ovary (CHO) K1 cells (A) and F5-5 mouse erythroid leukemia cells (B) were transfected with 300 ng of reporter construct p3TP-Lux, 100 ng of the internal control pRL-1PGK, and 200 ng of either control vector pactEF or Smad7 expression vector pactEF-Smad7 and then cultured in the absence or presence of 1 nmol/L activin. Transcriptional activity of the 3TP promoter was measured as firefly luciferase activity in the cell lysate. Values were normalized by comparison with values for sea pansy luciferase activity, which was driven by the constitutive promoter of the mouse phosphoglycerate kinase gene in pRL-1PGK. Results are expressed as the increase in induction (x-fold) compared with the value in the untreated vector control. Values are the mean and SD from at least 3 assays. (C) ST2 mouse fibroblast cells rendered highly responsive to activin by transfection of activin receptors in the p3TP-Lux assays. In parent cells, activin treatment produced only a weak activation of the 3TP promoter. ST2 cells were transiently transfected with 300 ng of p3TP-Lux, 100 ng of pRL-1PGK, and 50 ng each of expression vector for ActR-II (pactEF-ActR-II) and either ActR-I (pactEF-ActR-I) or ActR-IB (pactEF-ActR-IB) and then cultured in the absence or presence of activin. Luciferase activity was determined as described above. (D) Effects of transfection of activin receptors into NMuMG mouse mammary epithelial cells. Cells were treated and transcriptional activation was assessed as described in (C). (E and F) Efficient suppression by Smad7 of p3TP-Lux activation by activin in the cells producing ActR-I but only inefficient suppression in the cells producing ActR-IB. Increasing amounts of Smad7 vector (0-200 ng) were cotransfected with vectors for ActR-II and either ActR-I or ActR-IB (50 ng each) into ST2 cells (E) and NMuMG cells (F). After treatment with activin, the cells were harvested for the luciferase assay described above. (G) Immunoblot showing comparable levels of the type I receptor proteins, ActR-I and ActR-IB, in transiently transfected cells. NMuMG cells were transfected with carboxy-terminal Flag-tagged ActR-I or ActR-IB in the absence or presence of Smad7. A fixed amount (20 μg each) of total protein from the whole-cell lysate was analyzed by gel electrophoresis and subsequent immunoblotting with anti-Flag monoclonal antibody.

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