(A) Masson’s trichrome staining of the proximal aorta of control (Tbr1^+/+) and mutant (Tbr1^MR/+) mice at 20 weeks of age. Scale bars: 1,000 μm. The dashed rectangles indicate the areas used for in situ analyses. (B) Aortic diameters of mice of the indicated genotypes at 16 weeks of age. F, females (n = 13 for Tbr1^+/+ and n = 10 for Tbr1^MR/+); M, males (n = 12 for Tbr1^+/+; n = 6 for Tbr1^MR/+). No sex-specific differences were observed within any experimental group. P values refer to Kruskal-Wallis test with FDR-based multiple comparison correction. (C) Representative parasternal long-axis echocardiographic view showing the boundaries from which measurements were taken for aortic root (yellow arrows) and ascending aorta (blue arrows). (D) Representative immunofluorescence (IF) images of the aortic root of 16-week-old mice of the indicated genotypes probed with an anti–p-Smad2 or an unrelated anti–p-Smad2/3 antibody. Scale bars: 50 μm. Image enhancement for visual display was applied uniformly to all panels. Experiment was conducted at least 3 times. (E) Representative IF images of the aortic root and ascending aorta of 12-week-old mice probed with an anti–p-Smad2 antibody. Scale bars: 50 μm. Image enhancement for visual display was applied uniformly to all panels. Experiment was conducted at least 3 times. (F) Immunoblot of protein lysates from the aortic root and ascending aorta of mice of the indicated genotypes at 24 and 32 weeks of age after probing with antibodies that recognize p-Smad2 and β-actin (n = 3). Quantification of p-Smad2 after normalization to β-actin is shown on the right. P values refer to 1-way ANOVA followed by Holm-Sidak’s multiple comparisons test. Numerical data are presented as scatter dot-plots with boxes, with the box denoting the mean; error bars identify the 95% confidence interval.