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. 2004 Sep;6(5):584–594. doi: 10.1593/neo.04217

Figure 6.

Figure 6

Representative semiquantitative Western blot approach of ERβ 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) We clearly demonstrate that the ERβ expression at 53 kDa is decreased during estrus at the protein level in the uterus of all female offspring exposed to EE2 and the 0.1- and 50-mg dose of BPA compared with the negative control group. Within the 0.1- and 50-mg dose of BPA, we could detect only a very weak ERβ immunoband. Protein loading was normalized to β-actin using a monoclonal primary antibody at 1:15,000 dilution (Sigma), which was specific for a band at 42 kDa. (B) Additional control experiments investigated the specificity of the immunoreactions against ERβ at 53 kDa within the Western analysis by comparing two different commercial antibodies (PA1-311, Affinity Bioreagents, and GR39, Oncogene Research Products). Both antibodies raised against ERβ revealed the same specific immunobands in positive (uterus, liver, and heart) and negative (testis) tissue controls. (C) Statistical analysis of the semiquantitative Western blot approach of ERβ expression of uterine protein at estrus stage of all female Sprague-Dawley offspring. The optical density of the ERβ and β-actin immunobands was measured by integrating the average 8-bit gray-scale value of each immunoband. To standardize for differences in background intensity between Western blots, the background 8-bit gray scale value was subtracted from each immunoband average 8-bit gray-scale value. The amounts of the ERβ message per tissue sample of each case were expressed as the relative ERβ abundance normalized with that of β-actin expression (ratio ERβ/β-actin) and are reported as the mean ± SD. The statistical analysis revealed that the ERβ expression is decreased during estrus at the protein level in the uterus of all female offspring exposed to EE2 and the 0.1- and 50-mg dose of BPA compared with the negative control group.