Bradford reagent

Ten mg of freeze-dried ground material was homogenized in 1 ml extraction buffer containing 250 mM potassium phosphate (pH8.0), 0.5 mM EDTA, and 10 mM β-mercaptoethanol. The amount of soluble proteins in each sample was determined using Bradford reagent (Solarbio life sciences) according to the manufacturer’s instructions. Mean values from four biological replicates were obtained.

Total proteins extracted from JEG-3 cells or JEG-3-ORGs were lysed in lysis buffer containing a protease inhibitor mixture and quantified using Bradford reagent (Solarbio, China). The lysates were separated by SDS-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride membrane. The membranes were incubated overnight at 4°C with primary antibodies against human HLA-G (1,1,000, Abcam, UK), PAPPA2 (1,1,000, Abcam, UK), and β-tubulin (1,5,000, proteintech, China); they were incubated subsequently with horseradish peroxidase-conjugated rabbit or mouse secondary antibodies (1,10,000, Abcam, UK). After treatment with enhanced chemiluminescence reagent (Meilunbio, China), a ChemiDoc image analyzer was used for densitometric analysis. β-tubulin served as the loading control.