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. 2022 Jul 19;9:770065. doi: 10.3389/fcvm.2022.770065

FIGURE 1.

FIGURE 1

The klotho (Kl) genetic deletion leads to calcification in the aortic valve hinge and annulus with upregulation of Tgfb1 and TGFβ-dependent SMAD2 signaling in 10–12-week-old mice. (A–D) Alizarin red staining of wild-type (A) and Kl–/– (B–D) mice. Scale bars = (A–B) 200 μm; (C) 100 μm; (D) 50 μm. (E–I) Immunohistochemistry showing localized pSMAD2 levels in AV hinge and aortic annulus of wild-type (E,G) and Kl–/– (F,H). The pSMAD2-stained area was quantified using NIH Image J software (I). (J) qPCR study to quantify Tgfb1 expression in the pooled and micro-dissected tissue samples of AV and annulus from wild-type and Kl–/– mice. (K) Western blotting analyses showing levels of phosphorylated SMADs (p SMAD2, pSMAD3), total SMADs (SMAD2, SMAD3), and β-actin in micro-dissected and pooled tissue samples containing AV and annulus from wild-type and Kl–/– mice. (L) Densitometric analysis quantifying band intensities of western blots. The densitometry graphs of pSMAD2 and pSMAD3 were normalized with total or non-phosphorylated form of the proteins or β-actin. Values indicate mean ± SD, and significant “p-values” between wild-type and Kl–/– groups were given on the top of histograms.