Nuclease mix

Each fresh semen sample was prepared for protein analysis as described previously [6 (link)]. Seminal plasma, immature germ cells, and non-sperm cells were removed by centrifugation (350×g, for 20 min at room temperature) through 50% PureSperm (Nidacon, Mölndal, Sweden)/FertiCult medium (FertiPro N.V., Beernem, Belgium). The sperm pellet was washed once by resuspension in 1 mL of Tris-buffered saline (10 mM Tris-HCl pH 7.6, 100 mM NaCl) and further centrifugation (350×g, for 20 min at room temperature). The sperm pellet was then resuspended in 500 μL of lysis buffer containing 20 mM Tris, 2% SDS and 1% Nuclease Mix (GE Healthcare Life Sciences, Piscataway, NJ, USA). Lysates were sonicated on ice (40 Hz, 60 pulses) and centrifuged (14,000×g, for 20 min at 4 °C). The supernatants were recovered, and the pellets were discarded. The protein concentration was assayed according to Bradford’s method (BioRad, Hercules, CA, USA) by following the kit manufacturer’s instructions. Protein lysates were stored at − 80 °C prior to analysis.

Gills were chosen as the experimental tissue for comparing proteins abundance. Gills are directly in contact with water and responsible for oxygen extraction, gene chip studies have shown that bivalve tissues directly in contact with the external environment are more responsive to environmental perturbation than internal tissues (Clark et al., 2013 (link)). Hence, proteomics profiles of the gills can act as an effective proxy of the early whole-animal response.
Frozen gills were crushed as described above for the determination of ODH activity and arginine content. For each animal, 100 mg of the obtained powder was homogenized in 100 mM Tris–HCl (pH 6.8) with 1% of Protease inhibitor mix (GE Healthcare, Little Chalfont, UK), centrifuged (50 000 g, 5 min, 4 °C) and supernatants were transferred to new tubes. Nucleic acids were then removed (nuclease mix, GE Healthcare, following manufacturer’s instructions). Samples were precipitated at 4 °C using TCA 20% (1/1:v/v, overnight). After centrifugation (20 000 g, 30 min, 4 °C), pellets were washed with 70% acetone and re-suspended in Destreak buffer (GE Healthcare, Little Chalfont, UK) containing 1% ampholytes (IPG Buffer, pH 4–7; GE Healthcare, Little Chalfont, UK). Protein concentrations were determined using a modified Bradford assay (Ramagli, 1999 ), and all samples were adjusted to 200 µg of proteins in 250 µl.

Protein extracts of HeLa cells were obtained by adding 150µL of a RIPA buffer supplemented with a protease inhibitor cocktail (Sigma), Nuclease Mix (GE Healthcare), and a PhosStop phosphatase inhibitor cocktail (Roche) directly to 1 × 10⁶ cells. Extracts (20 µg/lane) were subjected to 4–12% SDS-PAGE and electroblotted to a nitrocellulose membrane (GE Healthcare). Membranes were blocked with 5% non-fat milk in PBS containing 0.1% tween 20 (PBST) for 1 h at RT. After two washes with PBST, the following antibodies were used: anti-cTXNPx antisera 1:6000 diluted in 1% Bovine Seroalbumin BSA-PBST for 1 h at RT, anti-pERK, and anti-ERK (Cell Signaling) diluted 1:1000 in 5% BSA-PBST overnight at 4 °C. After 3 washes, membranes were incubated with HRP conjugated anti-rabbit diluted 1:7000 in BSA-PBST for 1 h at RT. The signal was developed with SuperSignal^TM West Pico Chemiluminescent Substrate (Thermo Fisher Scientific Inc., Waltham, MA, USA).

The placenta and endometrium from 18 IUGR and 18 NBW (6/stage) were used to extract protein, as we described previously [3 (link)]. Briefly, approximately 0.2 g frozen samples were crushed to powder in liquid nitrogen, then homogenized in a lysis buffer containing 7 M urea, 2 M thiourea, 4% 3-[3-(-cholamidopropyl)-dimethylammonio]-1-propanesulfonate, and 50 mM dithiothreitol with protease inhibitors (GE Healthcare, Piscataway, NJ). An ultrasonicater (Sonics Model VC 750, Sonics and Materials, Newtown, CT) was set at 20% power output and used to break down the mixture for 10 min at 0°C. After the addition of 1% (vol/vol) nuclease mix (GE Healthcare), the mixture solution was kept at room temperature for 1 h to completely solubilize proteins, followed by re-sonification for 10 min as described above to thoroughly break down cell membranes. The homogenate was centrifuged for 10 min at 13,000 g at 4°C to settle down the insoluble components. The supernatant fluid was obtained and its protein concentration was determined using the Brandford method. Portions of the homogenate (1 mg of protein for both placenta and endometrium) were stored at -80°C.

The frozen tissues were rinsed in ice-cold PBS buffer and then placed in liquid nitrogen and ground thoroughly to a very fine powder. Tissue powder (100 mg) was dissolved in 500 μl of lysing solution containing 7 M urea, 2 M thiourea, 4% CHAPS, 40 mM DTT, 2% IPG buffer, pH 3-10 or pH 4-7, 1% Nuclease Mix and 1% Protease Inhibitor Mix (GE Healthcare, Amersham, UK), incubated for 2 h at room temperature with vortexing once every 15 min, and centrifuged at 15000×g for 1 h at 4 °C. The supernatant was collected and purified using a Plus One 2-D Clean-up kit (GE Healthcare, Amersham, UK). The concentration of each protein sample was determined using a Plus One 2-D Quant Kit (GE Healthcare). Protein samples were aliquoted and stored at -80 °C for 2-DE analysis.