Goat anti mouse and goat anti rabbit hrp conjugated igg

Placental primary cells were washed with HBSS and homogenized in RIPA Buffer (Sigma-Aldrich) supplemented with cOmplete™ Mini protease inhibitor (Roche, Mannheim, Germany). Total protein concentration was determined using the Lowry method; 20 μg of total protein were loaded on precast 10% Bis-Tris gel (NuPAGE™, Novex, Life Technologies). Proteins were blotted on a 0.45 μm nitrocellulose membrane (Hybond, Amersham Biosciences, GE Healthcare Life Sciences, Little Chalfont, UK) and blotting efficiency was determined with Ponceau staining (Ponceau S solution, Sigma-Aldrich). Primary antibodies were diluted as described in Supplementary Table 2 and incubated on membranes overnight at 4°C. HRP-conjugated goat anti-mouse and goat anti-rabbit IgG (1:5000, Bio-Rad) were used as secondary antibodies and incubated on membranes for 2 h at room temperature. Immunodetection was performed with a chemiluminescent immunodetection kit (WesternBright Chemilumineszenz Substrat für Film, Biozym) according to the manufacturer's instructions. Images were acquired with an iBright CL 1000 Imaging System (Thermo Fisher Scientific) and band densities were analysed with Image Studio Lite 5.2. Results are presented as a ratio of band densities of target protein and reference protein beta-actin.