Quantitative real-time RT-PCR and western blotting analysis
Extraction of total RNA of tissues and cells, quantitative real-time PCR and western blotting were performed as described previously (He et al., 2014). For quantitative real-time RT-PCR, total RNA of placental tissues and cells were extracted by TRIzol reagent (Invitrogen). 2 µg RNA was reverse transcribed to generate cDNA by superscript reverse transcriptase (Invitrogen). Quantitative real-time PCR was carried out using MiniOpticon™ Real-Time PCR Detection System (BioRad, Hercules, CA). Real-time PCR reaction solution consisted of 2.0 μl diluted cDNA, 0.2 μM of each paired primer and 1×PCR Master Mix (TaKaRa, Otsu, Japan). SYBR Green (Roche Ltd, Basel, Switzerland) was used as detection dye. Amplification of the housekeeping genes β-actin was measured for each sample as an internal control for sample loading and normalization. The primers used were listed as followings: sFlt1 (NM_001159920), sense: TTGGGACTGTGGGAAGAAAC; anti-sense: TTGGAGATCCGAGAGAAAACA. β-actin (NM_001101), sense: TGTGTTGGCGTACAGGTCTTTG; anti-sense: GGGAAATCGTGCGTGACATTAAG. The temperature range to detect the melting temperature of the PCR product was set from 60–95℃. The specificity of PCR products was examined by melting curve at the end of the amplification and subsequent sequencing. To determine the relative quantitation of gene expression for both sFlt1 and β-actin genes, the comparative Ct (threshold cycle) method with arithmetic formulae (2-ΔΔCt) was used.
For the western blotting analysis, 70 mg of tissues were homogenized in cold RIPA lysis buffer containing protease inhibitor cocktail tablet (Roche, Indianapolis, IN) and primary cultured cells were scraped off the plate in the presence of RIPA lysis buffer containing protease inhibitor cocktail tablet. The protein concentration was quantified by BCA kit. Then lysates were quickly sonified in ice bath, boiled 5 min at 100ºC, and stored at -80ºC. 30 µg of protein samples were separated by 10% SDS-PAGE and subsequently transferred to nitrocellulose membranes. After blockage in 5% skim milk powder in 0.1% Tris-buffered saline/Tween 20 (TBST), membranes were incubated with primary antibodies for 11β-HSD1 (Santa Cruz, Cat# sc-19259, RRID: AB_2119513, Clone ID: C-17), 11β-HSD2 (1:2000; Abcam, Cat# ab80317, RRID: AB_1658782C), a disintegrin and metalloproteinase (ADAM)17 (1:2000; Abcam, Cat# ab2051 RRID: AB_302796) , ADAM10 (1:2000; Abcam, Cat# ab124695 RRID:AB_10972023, Clone ID: EPR5622) and GAPDH(1:2000; Abcam, Cat# ab181602, RRID:AB_2630358, Clone ID: EPR16891) at 4 °C overnight. Then, the membrane was incubated with a secondary horseradish peroxidase-conjugated antibody (Proteintech Inc,WuHan,China) for 1 h at room temperature. Immunoreactive proteins were visualized using the enhanced chemiluminescence Western blotting detection system (Santa Cruz). The chemiluminiscent signal from the membranes was quantified by image J software. To control sampling errors, the ratio of band intensities to GAPDH was obtained to quantify the relative protein expression level.