Sc 539

Cells were lysed using RIPA Buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 1% SDS, and 50 mM Tris pH 8.0) containing protease inhibitor cocktail (Pierce Biotechnology, Rockford IL). The lysates were chilled on ice and agitated by vortex every ten minutes for one hour. Total protein concentration was measured by Bradford assay (Bio-Rad laboratories, Hercules, CA). A total amount of 10–40 μg protein per sample was resolved by electrophoresis on a 8% SDS-polyacrylamide gel and transferred to a PVDF membrane (Millipore Corporation, Bedford MA). Membranes were probed with primary polyclonal rabbit anti-PR antibody (sc-539, Santa Cruz biotechnologies, CA), polyclonal rabbit anti-ERα antibody (sc-543, Santa Cruz Biotechnologies, CA), mouse monoclonal anti-GAPDH antibody (sc-4472, Santa Cruz Biotechnologies, CA), rabbit polyclonal anti-HES1 (sc-25392, Santa Cruz Biotechnologies, CA) or mouse monoclonal anti-ELF-5(sc-376737, Santa Cruz Biotechnologies, CA). The blots were then probed with appropriate horseradish peroxidase conjugated secondary antibody (Vector Laboratories, MD). The protein bands were visualized using enhanced chemiluminescence reagent Hyglo Quick spray (Denville Scientific, South Plainfield, NJ) per the manufacturer's suggested protocol. Relative protein expression was determined by ImageJ (National Institutes of Health, USA).

Total protein was extracted from the uterus and the PECs using radioimmunoprecipitation assay lysis buffer supplemented with phenylmethanesulfonyl fluoride (Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s instructions and detected by the Bicinchoninic Acid Protein Assay Kit (Tiangen Biotech Co., Ltd., Beijing, China). Approximately 50 μg of protein was separated by electrophoresis on polyacrylamide gels and subsequently transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk powder for 1.5 h and incubated overnight at 4 °C with the following primary antibodies: anti-ER-α (1:1000; ab3575, Abcam, Shanghai, China), anti- ER-β (1:1000; ab3576, Abcam), anti-PR (1:200; sc-539, Santa Cruz Biotechnology, Shanghai, China), anti-GAPDH (1:5000; Abcam) and anti-actin (1:5000; Abcam). After incubation, the membranes were washed three times with TBST and incubated with anti-rabbit/mouse IgG antibody (1:2000; CWBIO, Beijing, China) for 2 h at 37 °C, followed by washing with TBST. Finally, the membranes were immersed in a high-sensitivity luminescence reagent (BeyoECL Plus; Beyotime), exposed to film using a FusionCapt Advance FX7 (Beijing Oriental Science and Technology Development Co., Ltd., Beijing, China) and analyzed using Ipp 6.0 (Image Pro-Plus 6.0; Media Cybernetics).

The following primary antibodies were obtained from Santa Cruz Biotechnology Inc., USA; GR(H-300): sc-8992, PR(C-20) (which detects PRA and B isoforms): sc-539, AR(441): sc-7305, GAPDH(0411): sc-47724, MR(MCR, H300): sc-11412, ERα(MC-20): sc-542. The flotillin-1 (610820) antibody was purchased from BD Transduction Laboratories (USA). The following secondary antibodies were obtained from Santa Cruz Biotechnology Inc., USA; anti-mouse: sc-2005, anti-goat: sc-2350 (used as IgG for the ChIP assay) and anti-rabbit: sc-2313. The ligands dexamethasone (DEX), MPA, P4, NET-A, NET, aldosterone (ALD) and mibolerone (MIB) were obtained from Sigma-Aldrich (South Africa). Human tumour necrosis factor α (TNFα) was obtained from Celtic Diagnostics (South Africa). Protease inhibitor cocktail tablets (EDTA-free) (cat #04693159001) were obtained from Roche (South Africa). Cycloheximide (CHX) was purchased from Sigma-Aldrich (South Africa).