Amersham ecl plus western blotting reagents

Total protein was extracted from 25 mg frozen placenta tissue, 1.5 × 10⁶ cultured trophoblast cells or 1.5 × 10⁶ endothelial cells. The protein lysates were prepared by homogenisation in buffer (10 mmol/l Tris pH 8, 130 mmol/l NaCl, 1% [vol./vol.] Triton X-100, 10 mmol/l sodium fluoride, 10 mmol/l sodium phosphate and 10 mmol/l sodium pyrophosphate) with protease inhibitors (P8340; Sigma-Aldridge, St Louis, MO, USA). Lysates were centrifuged at 20,000 g for 10 min at 4°C. Protein concentrations were measured with a BCA protein assay kit (Pierce, Carlsbad, CA, USA). Proteins were electrophoresed on a 7.5% SDS gel (Bio-Rad, Hercules, CA, USA), loaded with 100 μg total protein per well, and then transferred to a nitrocellulose filter (Invitrogen, Carlsbad, CA, USA). The membrane was blocked with 5% (wt/vol.) non-fat milk for 1 h, incubated with rabbit polyclonal TLR4 (H-80, 1:200; Santa Cruz) and β-actin (1:2000; Abcam, Cambridge, MA, USA) overnight then secondary antibodies (1:2000 and 1:6000) for 1 h. Amersham ECL Plus Western blotting Reagents (GE Healthcare, Aurora, OH, USA) was used for detection. All antibodies were diluted in PBS and validated by Precision Plus Protein Kaleidoscope Standards (BIO-RAD, Hercules, CA, USA). Densitometric data from autoradiograms were quantified by Image J (https://imagej.nih.gov/ij/download.html).

Total protein was extracted from 1.5 × 10⁶ cultured trophoblast cells or endothelial cells. The protein lysates were prepared as previously described [15 (link)]. Proteins were electrophoresed on a 4%−15% SDS gel (Bio-Rad, Hercules, CA, USA), loaded with 100 μg total protein per well, and then transferred to a nitrocellulose filter (Invitrogen). The membrane was blocked with 5% non-fat milk for 1h, incubated with Mouse monoclonal FABPpm (ab93928, 1:1000; Abcam) and β-actin (1:2000; Abcam) at 4°C overnight, then secondary antibodies for 1h. Amersham ECL Plus Western blotting Reagents (GE Healthcare, Aurora, OH, USA) was used for detection. All antibodies were diluted in 5% non-fat milk and size validated by protein standard (BIO-RAD). Densitometric data from autoradiograms were quantified by Image J (https://imagej.nih.gov/ij/download.html).