Monoclonal anti enos

Placental tissue, trophoblast, and cell line pellets were extracted using a modified radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris, 150-mM NaCl, 1% NP-40, pH 8.6) with protease inhibitor. The protein was loaded onto a 10% sodium dodecyl sulfate–polyacrylamide gel and transferred to a nitrocellulose membrane after electrophoresis. Blocking was carried out with PBS containing 5% skim milk and 0.2% Tween-20 for 1 h at room temperature. The membrane was incubated with primary antibodies overnight at 4 °C. After washing with PBS containing 0.2% Tween-20, the membrane was incubated with secondary antibodies for 1 h at room temperature. The band signal was developed by ECL plus reagents (GE Healthcare, Uppsala, Sweden) and scanned using a Typhoon 9400 scanner (GE Healthcare). The signal intensity was quantified via densitometry using ImageQuant TL v2005 software (GE Healthcare). The antibodies for Western blots were purchased from Santa-Cruz (polyclonal anti-PLC9, polyclonal anti-CBX7, polyclonal anti-PHF11, monoclonal anti-OAS2, polyclonal anti-OAS3, polyclonal anti-RNF121, monoclonal anti-NHERF1, monoclonal anti-KRT7, monoclonal anti-KRT18, monoclonal anti-COX5A, polyclonal anti-GAPDH, and monoclonal anti-β-actin antibody) and Abcam (polyclonal anti-INPPL1, monoclonal anti-eNOS, and polyclonal anti-IL-1β antibody).