Western blot

Placental tissues (5 mm × 5 mm × 5 mm) were obtained within 15 min after delivery, avoiding infarcted, necrotic, and calcified areas. Then, the tissues were washed with cold PBS to remove maternal and fetal blood. Placental tissues were snap frozen in liquid nitrogen for 10 min and were then stored at −80°C until use. Western blot analysis was performed following standard procedures. Placental tissues were weighed and homogenized in RIPA lysis buffer (100 mg tissue per milliliter) containing a protease inhibitor in a mini bead–based homogenizer for 2 min at 4,000 rpm. The homogenate was centrifuged at 130,000 g for 15 min at 4°C, and the supernatant was utilized as the total placenta homogenate. Protein concentrations were detected using the BCA protein assay kit (Thermo Fisher Scientific, Inc.). Total protein (30 µg/lane) was loaded into each well by SDS-PAGE using a 10% gel and then transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was then blocked with 5% nonfat dry milk for 1 h at room temperature. Following overnight incubation at 4°C with mouse anti-NQO1 antibodies (1:3,000, Cat. ab28947, Abcam) and rabbit anti-SRXN1 (1:1,000, Affinity Biosciences Cat# DF12028, RRID:AB_2844833). Subsequently, the membrane was incubated with the horseradish peroxidase–conjugated anti-mouse IgG and anti-rabbit IgG (1:1,000, Cell Signaling Technology, United States) secondary antibody for 2 h at room temperature and detected by chemiluminescence. β-Actin (1:3,000, cat. ab8226, Abcam) was used as a loading control.