Peroxidase conjugated anti β actin rabbit monoclonal antibody

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 was isolated from cell lysates obtained from HESCs incubated with E₂, or E₂ + ORG, or ETO, or LNG, or MPA, or DEX for 24 h using lysis buffer (Cell Signaling Inc., Danvers, MA, USA) containing a protease inhibitor cocktail. The protein concentration was determined by a detergent-compatible protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Then, 20 µg of total protein was diluted in 2× sample buffer (Bio-Rad) and then boiled for 5 min. Samples were then separated by 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (Bio-Rad) and transferred onto a nitrocellulose membrane (Bio-Rad). The membrane was blocked overnight with 10% non-fat dry milk in TBS-T and then incubated overnight at 4 °C with mouse anti-ZBTB16 monoclonal antibody (ThermoFisher, Waltham, MA, USA). Following washing in TBS-T, the membrane was incubated with peroxidase-conjugated anti-horse IgG at a 1/5000 dilution (Vector Labs). The signals were developed using a chemiluminescence kit (Amersham; GE Healthcare, Piscataway, NJ, USA). The membrane was then sequentially stripped and re-probed with peroxidase-conjugated anti-β-actin rabbit monoclonal antibody at 1/1000 dilution (Cell Signaling Inc.).