Nb 500 700

Total cellular protein was extracted using RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA) supplemented with the Protease and Phosphatase Inhibitor Cocktail (Roche Applied Science). For the placenta, pieces were dissected as described for RNA preparation but were snap frozen in liquid nitrogen and stored at −80 °C. Frozen tissues were thawed for a few minutes in pre-chilled RIPA buffer (1 ml per100 mg), homogenized with a Polytron (Kinematica), and sonicated twice for 1 min (pulsed 2 s on/2 s off). After centrifugation (13 000 rpm., 15 min), 20–40 μg aliquots of protein were separated on MiniProtean TGX precast gels (Biorad) and transferred to a nitrocellulose membrane (Protran, Whatman). The membrane was blocked for 1 h at room temperature(RT) in PBS containing 0.05% tween-20 and 5% non-fat dry milk, incubated overnight at 4 °C with primary antibodies, and for 1 h at RT with an HRP-conjugated secondary antibody. Fractions were detected by Western Lightning Plus ECL (Perkin Elmer, Waltham, MA, USA). This study used primary antibodies against EPAS1/HIF2A (NB 100-122), FIH1/HIF1AN (EPR3658, NBP1-40688), TBP (NB 500-700), and MUC1 (EP1024Y, NB110-57234) from Novus Biologicals (Littleton, CO, USA). ARNT (ab2771), EGLN2 (ab108980), and MUC1 (ab101352) were purchased from Abcam (Cambridge, MA, USA).

The Nuclear/Cytosol Fractionation Kit (BioVision) was used for nuclear fractionation. Extracted proteins were separated by running SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membrane. After the membrane was blocked in 5% fat-free milk in 1 × TBST and washed three times with 1 × TBST, it was immersed in 5% fat-free milk in 1 × TBST solution containing a primary antibody against HIF-2α (Novus, NB100-122; 1:200), Oct-4 (abcam, ab19857; 1 μg/ml), Nanog (ThermoFisher, PA1-097; 1:2000), Beta-tubulin (Novus, NB600-936; 1:1000), TATA binding protein (Novus, NB500-700; 1:1000), or Beta-actin (Novus, NB600-501; 1:5000), respectively at 4 °C overnight. After the membrane was washed three times with 1 × TBST, a secondary antibody against HRP-conjugated goat polyclonal anti-rabbit IgG (1:10000) or HRP-conjugated rabbit polyclonal anti-mouse IgG (1:10000) were added for 30 minutes at room temperature (RT). Finally, results were photographed by using LAS-3000 (FUJIFILM).