Ma3 1000

Around 10–15 nuclear subfractions from each of the control, early, and late preeclamptic placenta groups were pooled randomly within each group. The same procedures were used for the cytoplasmic subfraction and for total homogenates. The protein concentration was measured by fluorescence. The samples were loaded onto a 10% SDS-PAGE gel and separated under reducing conditions. Following this, they were transferred to a PVDF microporous membrane (Merck Millipore, Billerica, MA). In the next step, the membrane was incubated with 5% nonfat milk in TBST buffer (250 mM Tris-HCL pH 7.6, 1.5 M NaCl, 0.01% Tween) for one hour at room temperature, and then overnight at 4°C with a primary antibody diluted in 1% nonfat milk in TBST buffer: 1 : 500 for IKBKG (MA5-15155, Thermo Scientific), 1 : 500 for Lamin A/C (MA3-1000, Thermo Scientific), and 1 : 1000 for GAPDH (MA5-15738, Thermo Scientific). Following incubation, the membrane was washed in TBST buffer, incubated for one hour at room temperature with labeled alkaline phosphatase (A3562, Sigma-Aldrich; dilution 1 : 10000 in 1% nonfat milk in TBST) secondary antibody, and again washed. The analysis was then performed using an ECL detection kit according to the manufacturer's instructions. The optical density of bands was analyzed using the ImageJ software [26 ].

The GCs were harvested and washed once in phosphate-buffered saline (PBS), then lysed on ice for 30 min with radioimmunoprecipitation assay (RIPA) buffer (CST, 9806), and supplemented with 1% (v/v) protease inhibitor Cocktail (HY-K0010) and 1% (v/v) phosphatase inhibitors (Cocktail I, HY-K0021; Cocktail II, HY-K0022; and Cocktail III, HY-K0023), which were purchased from MCE (Shanghai, China). Western blotting was performed as described previously (Wu et al., 2019 (link); Yang et al., 2020a (link)). Protein concentrations were determined using a BCA protein assay kit (TransGen Biotech, Beijing, China). Equal amounts of proteins (15–50 μg/lane) were separated by SDS-PAGE (12% acrylamide running gel) and transferred to a nitrocellulose membrane (BioTrace^TM NT; Pall Corp., FL, United States). The following antibodies were used in this experiment: beta catenin (ab32572; Abcam), inhibin alpha (ab81234; Abcam), HSD17B1 (ab134193; Abcam), MIF (ab227073; Abcam), caspase6 (ab185645; Abcam), laminA/C (MA3-1000; Thermo), and p-laminA/C-S22 (13448; CST, Shanghai, China). The antibodies were diluted to the recommended ratio with Beyotime (P0256; Shanghai, China) diluent. The Western blotting images were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, United States).