Direct detect

Briefly, placental homogenates were centrifuged at 13,000 g for 10 min at 4 °C, the supernatant was collected, and the protein concentration was measured using a spectrometer (Direct detect, Millipore, USA). Thirty µg of total protein was loaded in Criterion™ TGX (Tris-Glycine eXtended) Stain-Free™ precast gels (Biorad, City, Country). The gel was activated with the ChemiDoc MP imager (Biorad) and transferred to polyvinylidene difluoride membranes (PVDF) in the turbo system Trans-Blot turbo transfer (Biorad). Thereafter, membranes were blocked and incubated with primary antibodies for progesterone receptor (PR), androgen receptor (AR), estrogen receptor (ER) α and β, glucocorticoid receptor (GR), CYP11A1, 17 βHSD2 and adiponectin receptor 2 (ADIPOR2) (Supplementary Table 1). The protein expression in each sample was normalized to total amount of loaded protein in each sample using Image Lab 5.0 (Biorad).

Six leaves from each sample (about 150 to 200 mg) were chopped into 2–3 mm pieces and placed into Precellys tubes CK28R with 1 mL of ice cold TCA-acetone precipitation buffer [10% TCA in acetone] and homogenised using Precellys for 45 seconds at 6500 rpm. The homogenised samples were placed into −20°C freezer for 1.5 hr and then centrifuged at 4500 g, 5°C for 45 min. The supernatant was discarded and the pellet mixed with 1 mL of ice cold acetone, left overnight at −20°C and then centrifuged at 4000 g, 5°C for 30 min and the supernatant removed. This acetone precipitation procedure was repeated three times.
To solubilize proteins, 400 μL of lysis buffer [50 mM HEPES, 2% SDS, pH 7.5, with protease inhibitor] was added to pellets, homogenised using Precellys at 6000 rpm for 20 sec, cooled on ice for 5 min, sonicated for 10 minutes, centrifuged at 12000 g for 10 minutes. This protein extraction procedure was repeated once. Supernatants from the two extractions were pooled.
The protein extraction supernatant was buffer exchanged using a 5KDa molecular weight cut-off spin filter (VS0212, VIVA Science) into iTRAQ sample buffer [0.25 M TEAB (triethylammonium bicarbonate), 0.05% SDS, pH 8.5]. A protein assay was carried out using Direct Detect (Millipore).

Protein samples (100 µl) with a concentration of 2 mg/ml were diluted to a final volume of 450 µl with 20% acetonitrile in water containing 1% acetic acid. Samples were concentrated 10-fold by centrifugation using 3 kDa Amicon ultrafiltration units (Millipore). This buffer exchange was repeated three times. Protein concentration was determined by infrared spectrometry (Direct Detect, Millipore) and adjusted to 2 mg/ml. Electrospray ionization mass spectrometry was performed with a LTQ Orbitrap mass spectrometer (LTQ Orbitrap Discovery, Thermo Scientific). The instrument’s built-in syringe pump delivered the sample at a flow rate of 1 µl/min. In the Orbitrap 100 full scan MS spectra were acquired in the m/z range 150–2,000 at a resolution of 30,000 and averaged. Data acquisition was carried out for 1 min. For charge state deconvolution spectra were exported to the MagTran software (version 1.02, [21] (link)). Protonated (MH+) molecular masses were calculated in the mass range 10,000–30,000 Da.

Rabbit polyclonal anti-BFT2 antibodies (L2-23) and mAbs from hybridoma culture supernatants were purified by affinity chromatography using a Protein G column (GE Healthcare). Briefly, a 1 ml Protein G column was equilibrated with 3 column volumes (CVs) of PBS. Hybridoma culture supernatant was adjusted to pH 7.0 by adding 10x PBS (Gibco) and then loaded on the column at 1 ml/min. IgG was eluted with 0.1 M glycine pH 2.0 and the pH of the eluate was adjusted to 7.0 with 0.5 M Tris (pH 9.0). The purified IgG was dialyzed against PBS overnight at 4°C, and the protein concentration quantified using Direct Detect (Millipore). Aliquots were stored at -20°C.