Protease inhibitor cocktail and phosphatase inhibitor cocktail

Endometrial tissues or cultured HESCs were lysed in RIPA lysis buffer (Biosharp, China) added with protease inhibitor cocktail and phosphatase inhibitor cocktail (MedChemExpress, USA) for 30 min on ice. The supernatant was collected after centrifugation at 12,000×g for 20 min. Protein concentration was assessed with a Pierce BCA protein assay kit (Thermo Scientific, USA). The proteins were fractionated with SDS-PAGE gels, transferred to PVDF membranes (Bio-Rad, USA), and then blocked in 5% nonfat milk (Bio-Rad, USA) at room temperature. The membranes were incubated with appropriate primary antibodies overnight at 4 °C and then hybridized with a horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The bands were visualized with ECL solution (BioRad, USA). The antibodies used are summarized in Supplementary Table S3.

Protein extraction and Western blot analysis were performed as described previously (Gao et al., 2019 (link)). Tissues or cultured cells were lysed in RIPA buffer (Solarbio, Beijing, China) and a protease inhibitor cocktail and phosphatase inhibitor cocktail (MedChemExpress, NJ, United States). The samples were kept on ice for 30 min and then centrifuged at 12,000 g for 25 min at 4°C. After the protein concentration was determined, protein lysates were separated by SDS-PAGE and then transferred to PVDF membranes (Millipore, MA, United States), blocked with 5% skim milk or 5% BSA in Tris-buffered saline with 0.1% Tween 20 (TBST) for 2 h at room temperature, and incubated overnight with primary antibodies. After incubation with primary antibodies, the membranes were washed in TBST three times and then incubated with their specific HRP-linked secondary antibodies (1:5,000; Jackson ImmunoResearch, PA, United States) in TBST for at least 1 h at room temperature. Bands on the membranes were visualized by ECL Western Blotting Substrate (Solarbio) using the Bio-Rad ChemiDoc^TM XRS + System. The related antibody information is summarized in Supplementary Table 5.

Tissues or cells were lysed in lysis buffer (Biosharp, China) supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail (MedChemExpress, USA) and clarified by centrifugation at 12,000 × g for 15 min. The protein concentrations were determined using a Pierce BCA protein assay kit (Thermo Scientific, USA). Protein samples (30 μg) were fractionated with SDS-PAGE, transferred to PVDF membranes (Bio-Rad, USA), and incubated in 5% nonfat milk (Bio-Rad, USA) at room temperature. The membranes were incubated with primary antibodies at 4 °C overnight and were then incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. The signals were visualized with ECL solution (Bio-Rad, USA) and quantified by analysis of the integrated density normalized to the level of β-actin using ImageJ. The antibodies used are shown in Supplementary Table S2.