Image quant tl v2005

1-methyl-7-nitroisatoic anhydride (1M7) was synthesized according to the method described by Turner and co-authors^{57 (link)}. Empty puromycin treated salt-washed ribosomes were prepared as described previously⁵⁸ . The ribosomes were treated with 1M7 at 30°C for 20 min^{59 (link); 60 (link)}. Ribosomes were precipitated, pelleted and RNA was isolated by SDS-acidic phenol-chloroform treatment^{53 (link)}. Primer extension reactions were performed as described previously^{61 (link)}. Oligonucleotide primers used for the primer extension analysis are listed in Supplemental Table S4. All assays were performed with two independent biological replicates. SHAPE reactivities were quantified using Image Quant TL v2005 software (GE Healthcare). Reactivities for the analysed nucleotides were normalized by dividing by the average reactivity of all nucleotides. Absolute reactivities were calculated by subtracting the non-reagent intensities from the 1M7-modified intensities. The reactivities that were less than zero were reset to zero. Representative results of SHAPE analysis are shown in Figure S2.

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).