Tissue extraction buffer

Pyloric tissues were stored at -80 °C until use. Pyloric tissue was homogenized in tissue extraction buffer (Thermofisher Scientific, Waltham, MA). The tissue lysates were then centrifuged for 15 min at 15,000 × g and the protein concentration of the supernatant was determined using a protein assay kit (Thermofisher Scientific). Equal amounts of tissue proteins were then separated by 10% SDS-PAGE gel and transferred onto a nitrocellulose membrane. After blocking the membranes with 5% non-fat milk diluted in buffer (10 mM Tris-HCl, 100 mM NaCl and 0.1% Tween 20) for 3 h at room temperature, specific proteins were probed with primary antibodies (anti-mouse ERα mAb (1:1,000 dilution), anti-rabbit AR pAb (1:100 dilution) or anti-mouse ERβ mAb (1:1,000 dilution)) for 4 h at room temperature. Actin antibody was used as a control and run on a separate gel from the target proteins due to image signal interference; IR-dye conjugated secondary antibodies (anti-mouse IRDye800 or anti-rabbit IRDye800) at a dilution of 1:20,000 were then added and incubated for 1 h at room temperature.

Caspase-3 and nuclear factor-kappa B (NF-κB; p65) protein expression were analyzed by western blots to determine involvement of apoptosis and inflammation in neuroprotection. Cleaved caspase-3 is the biologically active form of intact caspase-3 and induces apoptosis. Brain tissue was homogenized in ice-cold tissue extraction buffer (Invitrogen, containing 1% protease inhibitor cocktail. Samples containing 20 μg protein were loaded into each well of NuPAGE precast 8–16% Bis-Tris gels (Invitrogen) (Lin et al., 2011). After electrophoresis, proteins were transferred to nitrocellulose membranes (Invitrogen). Membranes were blocked in NuPAGE blocking buffer (Invitrogen) and then incubated with primary antibodies, mouse anti-monoclonal antibody β-actin (1:10,000), NF-κB (p65) (1:5,000), and caspase-3 active form (1:5,000) (all Invitrogen), at room temperature for 2 hours. After washing, membranes were incubated with horseradish peroxidase-conjugated sheep anti-mouse antibody at room temperature for 1 hour and processed using ECL Western blotting detection reagents (GE Healthcare, Piscataway, NJ, USA). Protein bands were then imaged (Kodak Image Station 179 4000R station, NY, USA).