Figure 6.

TGFβ3 is required for TGFβ-SMAD cardiovascular pathway activation. (A–C), Western blot analysis of fetal hearts and the accompanied ascending aorta (E18.5) shows protein levels of the phosphorylated SMADs (pSMAD2, pSMAD3, pSMAD1/5) and total SMADs (SMAD2, SMAD3, SMAD1/5). (D) A common and independent β-actin blot (C, bottom) was used for normalizing data from pSMAD2, pSMAD3, and pSMAD1/5 blots. There was no difference in the β-actin levels between any cardiovascular tissues taken from wildtype and Tgfb3^−/− mice. Densitometric quantification of phosphorylated proteins after normalization to β-actin and total non-phosphorylated proteins is shown on the right (A–C). E, Representative western blots of pooled samples (3 hearts and aortas/sample) from wildtype, Tgfb3^+/−, and Tgfb3^−/− fetuses (E14.5) for pSMAD2, SMAD2, and β-actin. Each western blot was repeated three times with similar results. Western blots and densitometric quantification show the levels of pSMAD2 (60 kDa) and SMAD2 (60 kDa) (A,D), pSMAD3 (52 kDa) and SMAD3 (52 kDa) (B), pSMAD1/5 (60 kDa) and SMAD1/5 (60 kDa) (C), and β-actin (42 kDa) (A–D). Note that levels of pSMAD2, pSMAD3, and pSMAD1/5 are increased in individual samples taken from TGFβ3-deficient mice (A–C). The levels of pSMAD2 were also higher in pooled samples of both Tgfb3^+/− and Tgfb3^−/− fetuses compared to pooled sample from wildtype fetuses. Tgfb3^−/− had slightly higher levels of pSMAD2 than the Tgfb3^+/− fetuses. Data points excluded from statistical analysis (Student’s t test) are indicated by grey symbols (A–C). Individual densitometric values from each pooled samples were plotted (E). All western blots (A–C) with individual samples were done in triplicate. Thus, all data points represent an average values of three independent blots (A–C). p-values are shown in the figure. Numerical data from multiple individual samples are presented as scatter dot-plots with boxes denoting the mean; error bars indicate the SEM.