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. Author manuscript; available in PMC: 2017 Jul 1.
Published in final edited form as: Arterioscler Thromb Vasc Biol. 2016 May 19;36(7):1398–1405. doi: 10.1161/ATVBAHA.116.307526

Figure 2. pSmad1/5/8 activation precedes and later localizes with calcific nodules in Klotho−/− AoV.

Figure 2

AoV calcification in Klotho−/− mice (I–L) compared to WT littermate controls (A–D) was examined at 2, 4, 6 and 8 weeks of age. Evidence of calcification is first observed at 6 weeks and persists through 8 weeks of age, as detected by von Kossa staining (K–L, arrowheads). pSmad1/5/8 (green) activation precedes calcification at 4 weeks (N, white arrowheads), and then localizes with calcific nodules at 6–8 weeks (O–P) in Klotho−/− AoV. Smooth muscle cells of the aorta are detected by AlphaSMA staining (red) in Klotho−/− and control AoV (E–H, M–P). At least 3 hearts were analyzed for each phenotype and time-point. Western blot analysis of phosphoSmad1/5/8, total Smad1/5/8 and GAPDH protein levels in Klotho−/− AoV compared to WT littermate control (Q). Quantification of phosphoSmad1/5/8 levels normalized to total Smads and GAPDH (R); n=3 was used for each Klotho−/− and control group in which each n is equal to 10–12 AoV. Error bars are shown as mean ±SEM and scatter plot. Statistical significance was determined by unpaired t-test with Welch’s correction (p< 0.05).