Figure 4.

Representative Western blot analyses of ERα expression of uterine protein at estrus stage of female Sprague-Dawley offspring exposed to 17α-ethinyl estradiol and bisphenol A in utero. Gravid dams were fed by gavage on gestation days 6 through 21 with either 2% cornstarch (negative control; CO) at 10 ml/kg per day, 0.2 mg/kg per day EE2 (EE2), used as a positive control, or 0.1 mg/kg per day BPA (BPA0.1) or 50 mg/kg per day BPA (BPA50). The female offspring were then sacrificed in estrus at 4 months of age. (A) The full-length ERα expression at 64 kDa is increased in all female offspring exposed to EE2 and the 50-mg dose of BPA compared with the negative control group. Within the 0.1-mg dose of BPA and the control group only very weak but specific ERα immunobands of the full-length ERα variant at 64 kDa could be detected. Only two immunoreactive bands at 56 and 42 kDa from homogenates of rat uteri from all treated animals showed strong staining. Protein loading was normalized to β-actin using a monoclonal primary antibody at a 1:15,000 dilution (Sigma), which was specific for a band at 42 kDa. The anti-ERα antibody specifically reacted with three bands at 64, 56, and 42 kDa from homogenates of rat uteri. (B) Binding to all immunopositive bands was eliminated when the antibody was preincubated with antigen Erα peptide. (C) Substituting TBS containing 0.5% NFDM (∅ 1.Ab) instead of primary antibody (+ 1.Ab) for ERα led to no more immunoreactivity. Protein, in the amounts of 14, 30, and 15 µg, was loaded.