Expression of BMP2, RUNX2, DKK1, LRP6, β-catenin, OPN, VCAM-1, p-ERK1/2, ERK1/2 and Ki-67 in aortic root, BMP2, RUNX2, p-ERK1/2 and ERK1/2 in thoracic artery, and β-catenin, RUNX2, BMP2 and ALP in HASMCs were determined by immunofluorescent staining of the 5-µm frozen sections or the slides of cell culture and primary antibodies. The negative control immunofluorescent staining was conducted using normal IgG to replace primary antibody. After the images were captured, the mean fluorescent intensity (MFI) of immunofluorescent image was calculated as described 37. Expression of target proteins were determined by Western blot with total or nuclear proteins by Western blot as described 38.

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