*4.3. Western Blot Analysis*

For protein expression analysis, placental tissue of rat NP/LP (4 dams with 2 pups/dam) and mouse E18.5 eNOS−/− versus C57BL/6 control (8 dams with 1 pup each) was homogenized by mincing in 20 mL RIPA bu ffer, consisting of 50 mM Tris (pH 7.2), 10 mM EDTA, 150 mM NaCl, 0.1% SDS, 1.0% Triton X-100, 1.0% sodium deoxycholate, 20 μL/mL proteinase inhibitor (Complete proteinase inhibitor, Santa Cruz Biotechnology Inc., Dallas, TX, USA), and 2 mM Na3VO4. Bu ffer amount was adjusted to sample weight. The protein concentration was determined by the kit (Pierce, Rockford, IL, USA). Rat samples containing 30 μg/<sup>44</sup> μl and mouse samples containing 30 μg/40 μL of protein were boiled at 95 ◦C for 8 min and separated on a 10% denaturing SDS-PAGE gel (for Rarres1 measurements of rat samples, 12% gel was used). Semi-dry electro-blotting was performed using Hartenstein GB33 PVDF membranes (Bio-Rad Laboratories, Hercules, USA), which were then blocked with Rotiblock (Roth, Karlsruhe, Germany) for 60 min. The membrane was incubated overnight at 4 ◦C with a polyclonal rabbit anti-rat antibody to Rarres1 (Biorbyt, Cambridge, UK) at a concentration of 1:250, or polyclonal rabbit anti-rat antibody to Rarres2 (Thermo Fisher, Waltham, MA, USA) at a concentration of 1:500 (rat)/1:1000 (mouse). Subsequently, the membrane was incubated for 60 min at room temperature with a secondary donkey anti-rabbit antibody (GE Healthcare, Amersham, UK) in the concentration 1:10,000 (for Rarres1 rat–blots 5% milk powder was added). As a reference, a monoclonal mouse anti-vinculin antibody at a concentration of 1:2000 and a monoclonal mouse anti-β-Tubulin antibody at a concentration of 1:10,000 (both from Sigma Aldrich, St. Louis, MO, USA) followed by a secondary sheep anti-mouse antibody (GE Healthcare, Amersham, UK) were used. As both reference genes resulted in similar results, only β-Tubulin blots were displayed. Immunoreactivity was visualized using the fluorescent ECL Plus Western Blotting Substrate according to the manufacturer's instructions (Thermo Fisher Scientific, Waltham, MA, USA) and quantified with a luminescent imager (LAS-1000, Fujifilm, Berlin, Germany) and AIDA Image Analysis software (version 2.1, Elysia-raytest GmbH, Straubenhardt Germany).

Coomassie Brilliant Blue staining served as the loading control.
