Peroxidase conjugated anti goat igg

To evaluate FKBP51 protein levels, cell lysates from HESCs cultured for 24h with E₂ or E₂+P4 or E₂+MPA or E₂+ETO were diluted in 2X sample buffer (Bio-Rad, Hercules, CA) and then boiled for 5 minutes. The cell lysates were subjected to reducing SDS-PAGE on a 10% Tris-HCl gel (Bio-Rad), with subsequent electroblotting transfer onto a 0.45-μm nitrocellulose membrane (Bio-Rad). After transfer, the membranes were blocked overnight in Tris-Buffered Saline (TBS) with 10% non-fat dry milk and then incubated with goat anti-FKBP51 polyclonal antibody at 1/1000 dilution (R&D Systems) or with rabbit anti-total (T) and phosphorylated (P)-AKT, or with rabbit anti-T and P-ERK1/2 MAPK antibodies (Cell Signaling) at 1/1000 dilution for primary antibody labeling. Membranes were rinsed in TBS-T (TBS with 0.1% Tween 20) subsequently incubated with peroxidase-conjugated anti-goat IgG at 1/5000 dilution (Vector Labs) and signals were developed using a chemiluminescence kit (Amersham; GE Healthcare, Piscataway, NJ). The membranes was sequentially stripped and re-probed with peroxidase-conjugated anti-β-actin rabbit monoclonal antibody at 1/1000 dilution (Cell Signaling Technology).

Total protein from BeWo cell samples was extracted with the RIPA lysis buffer (Sigma-Aldrich Co., St Louis, MO, USA) containing Complete Mini Protease Inhibitor Cocktail Tablets (Roche, Indianapolis, IN, USA). Protein concentrations were determined with the Quick Start Bradford Protein Assay (Bio-Rad, Hercules, CA, USA). Twenty micrograms of total protein from each sample were electrophoresed on 4–12% SDS-PAGE gels (Life Technologies Corporation), and electro-transferred onto nitrocellulose membranes (Bio-Rad). Membranes were probed with goat anti-human Flt-1 polyclonal antibody (AF321, R&D Systems, Inc., Minneapolis, MN, USA) in a 1:2,000 dilution at 4C° for 16h, and then with peroxidase-conjugated anti-goat IgG (Vector Laboratories, Burlingame, CA, USA) in a 1:5,000 dilution at room temperature for 1h. Protein bands were developed using the ChemiGlow Western Blotting Detection Reagents (Protein Simple, Santa Clara, CA, USA), and then scanned and imaged with a Fujifilm LAS-4000 Image Reader (GE Healthcare, Piscataway, NJ, USA).