Quantification of Aortic ALP Activity

ALP activity was measured by using an ALP assay kit (Nanjing Jiancheng Bioengineering, Nanjing, China). The proteins were extracted from the aortic tissues or A7r5 cells in 0.05% Triton X-100 in PBS and quantified using a bicinchoninic acid (BCA, ThermoFisher, Waltham, MA, USA) protein assay. The 5 μL supernatant of samples was mixed with reaction mixture including alkaline buffer solution 50 μL mainly containing disodium phenyl phosphate and substrate solution 50 μL mainly containing 4-aminoantipyrine and potassium cyanide. They were incubated at 37 °C for 15 min, then developer 150 μL was added into each well. The absorbance was detected at 520 nm wavelength and the results were normalized to the level of total protein.