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Due to the requirement for Tβ4 in VSMC differentiation (8, 9, 23), we assessed the phenotype of medial layer VSMCs by measuring expression of established contractile (αSMA and SM22α) and so-called synthetic markers ( Tropomysin and low-molecular-weight isoforms of Caldesmon) (24) by quantitative reverse transcription real-time PCR (qRT-PCR), immunofluorescence, and Western blotting (Figure 1, F–H

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