Beyoecl moon ecl kit

Primary GMECs and GMEC-line cells were collected and lysed in RIPA buffer containing PMSF (1 mM) at 4°C for 30 min, followed by centrifugation at 12,000 g for 10 min to collect the supernatant. Proteins were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% skim milk powder for 90 min and incubated with anti-ERα (1:100, Abcam), anti-PR (1:1,000, Abcam), anti-PRLR (1:300, Abcam), anti-cleaved caspase 3 (1:1,000, Abcam), anti-CSN2 (1:1,000, Abcam), and anti-GAPDH (1:10,000, Abcam) polyclonal antibodies overnight at 4°C. After three washes with TBST, the membrane was incubated with goat anti-rabbit IgG (H&L) secondary antibodies for 50 min at room temperature, followed by another three washes with TBST. Finally, the membrane was covered with BeyoECL Moon ECL Kit (Beyotime), and its chemiluminescence was detected using a chemiluminescent detector.

Both primary PECs and the PEC cell line were collected using 0.25% trypsin, and then centrifuged at 800 g to remove trypsin. After two washes with PBS, these cells were resuspended with RIPA buffer containing PMSF (1 mM) complete protease inhibitors. Cells were lysed in an ice-bath for 30 min and centrifuged at 10500 g for 10 min to collect the supernatant. The concentration of total protein was determined using a BCA Protein Assay Kit. Proteins were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% skim milk powder for an hour and incubated with anti-E2α (1:1000, Abcam), anti-PR (1:1000, Abcam) and anti-GAPDH (1:10000, Abcam) polyclonal antibodies overnight at 4 °C. After three washes with TBST, the membrane was incubated with specific goat anti-rabbit IgG (H&L) secondary antibodies for 1 h at room temperature, and then washed with TBST thrice. Finally, it was covered with BeyoECL Moon ECL Kit (Beyotime), and its chemiluminescence was detected using Chemiluminescent Detector.