SERCA2 Activity Assay

SERCA2 is a member of the ATPase family, which can decompose ATP into ADP and inorganic phosphorus. Analysis of the amount of inorganic phosphorus determined the level of ATPase activity by using ultramicro-Ca2+-ATPase detection kit (Jiancheng Bioengineering Institute, China). The treated hPVSMCs were digested, centrifuged, and the supernatant was removed, leaving layers of cells, and 200 μl of ultrapure water was added to each tube to prepare a 107/ml cell suspension, which was disrupted by an ultrasonic pulverizer. The prepared cell suspension did not centrifuge and the total protein concentration was measured by the BCA method. In vivo, pulmonary veins were isolated from WT and Tgm2−/− mice under hypoxia and normoxia. In brief, 9 vol of ultrapure water were added to the veins, and veins were mechanically disrupted in an ice bath, and centrifuged at 2,500 rpm for 10 min. Next, the supernatant was collected, diluted 10-fold, and the total protein concentration was measured by the BCA method. Enzymatic reactions and phosphorus determination were carried out according to the kit instructions. An UV spectrophotometer (Mapada UV-3100PC, China) was used to measure the absorbance of samples at a wavelength of 636 nm and 1 cm optical path after ultrapure water zero setting. The optical density (OD) value was used in the following formula to calculate the activity of ATPase. Activity of ATPase (U/mgprot) = (the OD value of the sample − the OD value of the control)/(OD value of standard-OD value of blank)×0.02 μmol/l×6×2.8÷total protein concentration of sample (mg protein/ml) (Guo et al., 2017).