Western blot analysis of Estrogen Receptor (ER) was performed by loading the indicated amounts of recombinant full-length human ER alpha (Product # A15674) and ER beta (Product # A15664) proteins, and 10 µL of PageRuler Prestained Protein Ladder (Product # 26616) per well onto a Novex® 4-20% Tris-Glycine polyacrylamide gel. Proteins were transferred to a PVDF membrane (Product # 88518) using the G2 Blotter (Product # 62288), and blocked with 5% milk in TBST for at least 1 hour at room temperature. ER alpha was detected at ~66 kD (left panel) using an ER monoclonal antibody (Product # MA5-12328) at a dilution of 1:200 in blocking buffer overnight at 4C on a rocking platform, followed by an HRP-conjugated goat anti-mouse IgG secondary antibody (Product # 31430) at a dilution of 1:40,000. Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34080). The blot was then stripped with Restore Plus Western blot stripping buffer (Product # 46430) for at least 15 minutes at room temperature, re-blocked with 5% milk in TBST for at least 1 hour at room temperature, and re-probed with an ER beta polyclonal antibody (Product # PA1-313, right panel) at a dilution of 1:2000 overnight at 4C, followed by an HRP-conjugated goat anti-rabbit IgG secondary antibody (Product # 31460) at a dilution of 1:40,000. Chemiluminescent detection was again performed using SuperSignal West Pico (Product # 34080).