Fig. 5.
TGF-β1 prolongs BMP-2 signaling and reduces the expression of the BMP-2 antagonist noggin. a Immunoblots of phospho-Smad1/5/8 (pSmad1/5/8) and phospho-Smad2 (pSmad2) proteins in whole-cell extracts of ST2 cells that were either untreated or treated with recombinant TGF-β1 (5 ng·ml−1), BMP-2 (100 ng·mL-1) or both growth factors. Cell lysates were collected on two consecutive days after the treatment. Anti-vinculin served as a loading control. b, c Densitometric analyses of the immunoblots shown in a. pSmad1/5/8 (b) and pSmad2 (c) protein levels are normalized to the vinculin loading controls. Mean± standard deviations were obtained from three independent experiments, and significant differences to either untreated cells or between identically treated cells on day 1 and 2 after the stimulation (***P < 0.001, **P < 0.01) are shown. d Effect of TGF-β1 on BMP-2-induced noggin expression in ST2 cells. Cells were treated with increasing concentrations (0.0, 0.5, 2.5, and 5.0 ng·mL-1) of TGF-β1 in the absence (−) or presence (+) of 100 ng·mL-1 BMP-2 for 24 h before total RNA was extractedand Nog mRNA levels were analyzed by qRT-PCR. Values normalized to Gapdh are expressed relative to the values of untreated cells. Mean± standard deviations were obtained from three independent experiments, and significant differences to either untreated cells or cells treated with BMP-2 alone (***P < 0.001) are shown