Nitrocellulose filter

Purified RNA was transfected into BHK21 cells via electroporation, 3 μg per 200 μl of 10⁶ BHK21 cells. Fourteen μl of clarified supernatant was fractionated on 4–12% SDS-PAGE and blotted onto nitrocellulose filters (Invitrogen, CA). HIV Env bands were detected by monoclonal antibody (mAb) 16H3 (Gao et al., 2009 (link)) followed by goat anti-mouse IgG-HRP (Sigma, Cat# A8924-.5ML). Env bands were developed with Pierce ECL Western Blotting Substrate (Thermo Scientific, Cat#. 32106). Yellow fever viral envelopes were fractionized in SDS-PAGE and detected by Dengue mAb 4G2 (Henchal et al., 1982 (link)) under non-reducing conditions and then conjugated with goat anti-mouse IgG-AP (Sigma, Cat # A3652-.5ML). Yellow fever viral envelope bands were developed with Western Blue Stabilized Substrate for Alkaline Phosphatase (Promega Cat# S3841).

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).