Western Blot Analysis of ERα and ERβ

Uterus and vagina were resuspended in lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 5 mM EDTA, 0.1% sodium dodecyl sulfate (SDS), 0.5% NP-40) containing 10 mM phenylmethylsulfonyl fluoride (PMSF) and 2 mg/mL aprotinin. The protein was obtained to detect the levels of ERα and ERβ in target tissue by western blotting. The western blot protocol and semi-quantitative analysis were carried out as described [42 (link)]. The antibody of rabbit anti-ERα polyclonal antibody (dilution 1/200, Santa cruz Biotechnology) or mouse anti-ERβ monoclonal antibody (dilution 1/1,000, Abcam Biotechnology) was used. All experiments were done in triplicate. The relative quantity of each antibody was measured by Alpha Ease FC (Fluorchem FC2) software. The density ratio of protein to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (dilution 1/1,000, Cell Signaling Technology) was calculated from the band density.