Figure 1: Structure of the Estrogen Receptor beta target protein.

Estrogen Receptor beta Target Introduction

Protein Function

• Estrogen Receptor beta (ERβ) is a member of the nuclear hormone receptor family NR3 subfamily, and its affinity for binding to estrogen is similar to ERα. It activates the expression of reporter genes containing estrogen response elements (EREs) in an estrogen-dependent manner.
• Human ERβ has multiple isoforms, among which isoform 1 (ERβ1) forms functional homodimers and heterodimers with other isoforms. After forming heterodimers with ERα, ERβ1 inhibits the signaling pathway of ERα.
• Various epigenetic factors, including high methylation of CpG islands, post-translational modifications of histones, and the influence of small non-coding RNAs or miRNAs, regulate ERβ.
• ERβ plays an essential role in tumor cell metabolism, involving pathways such as lipid metabolism, oxidative phosphorylation, and glycolysis.
• Clinical studies on prostate cancer, breast cancer, and thyroid cancer have shown that the ratio of ERα/ERβ changes during tumor development, with upregulation of the former and downregulation of the latter. Due to the increasingly evident anti-proliferative activity of ERβ, this receptor is expected to become an essential target for cancer therapy.

Protein Expression

• Isoform 1 in humans: expressed in the testes and ovaries, with lower expression levels in the heart, brain, placenta, liver, skeletal muscle, spleen, thymus, prostate, colon, bone marrow, breast, and uterus. It is found in breast cancer and ovarian cancer cell lines but not in colon cancer and liver cancer.
• Isoform 2 in humans: expressed in the spleen, thymus, testes, and ovaries, with lower expression levels in skeletal muscle, prostate, colon, small intestine, leukocytes, bone marrow, breast, and uterus.
• Isoform 4 in humans: expressed in the testes.
• Isoform 5 in humans: expressed in the testes, with lower expression levels in the spleen, thymus, ovaries, breast, and uterus.
• Isoform 6 in humans: expressed in the testes, placenta, skeletal muscle, spleen, and leukocytes, with lower expression levels in the heart, lungs, liver, kidneys, pancreas, thymus, prostate, colon, small intestine, bone marrow, breast, and uterus. Not expressed in the brain.

Protein Localization

• Nucleus

Image 2: ICC experimental result of Estrogen Receptor beta protein, using Anti-Estrogen Receptor beta (phospho S105) antibody (ab62257). Green: Estrogen Receptor beta.

Isoforms & post-translation modifications

• Human (Q92731):
  Isoforms 1-2, 4-6, 9 (Q92731-1~2, Q92731-4~6, Q92731-9) 52.9-59.2 kDa (predicted)
  Isoform 7 (Q92731-7) 48.9 kDa (predicted)
  Isoform 8 (Q92731-8) 41.9 kDa (predicted)
  Isoform 3 (Q92731-3) 35.9 kDa (predicted)
• Mouse (O08537):
  Isoforms 1-2 (O08537-1~2) 59.0-61.0 kDa (predicted)
  Isoform 3 (O08537-4) 48.5 kDa (predicted)
  Isoform 5 (O08537-7) 42.1 kDa (predicted)
  Isoform 4 (O08537-6) 38.1 kDa (predicted)
• Rat (Q62986):
  Isoforms 1-2 (Q62986-1~2) 59.1-61.1 kDa (predicted)
  Isoforms 3-4 (Q62986-3~4) 54.6-56.6 kDa (predicted)
  Isoform 5 (Q62986-5) 47.9 kDa (predicted)
• Phosphorylation

WB experiment tips

Precautions

• Please note that there may be differences in the expression levels of ERβ in different samples, for example, the expression of this protein may not be detected in breast cancer cells. Therefore, it is necessary to confirm the expression level of the target protein before the experiment. We recommend using the samples used in the antibody product manual as positive controls.
• ERβ is located in the cell nucleus, and ultrasonic disruption of the sample can help enrich more target proteins. To ensure sufficient loading amount, you can try to extract the nuclear fraction for WB experiment.
• ERβ contains multiple isoforms in humans, rats, and mice, so multiple bands may be detected in WB experiments.

Positive control

• Human MCF7 cell nuclear lysate

Negative control (no expression or weak expression)

• Human Hs 578T whole cell lysate

Example of results

Figure 3: WB experiment results of Estrogen Receptor beta protein, Anti-Estrogen Receptor beta antibody (ab3576).

Lane 1: Human MCF7 whole cell lysate (20 µg).
Lane 2: Human MCF7 nuclear lysate (20 µg).
Lane 3: Human T47D whole cell lysate (20 µg).
Lane 4: Human T47D nuclear lysate (20 µg).
Lane 5: Human SK-BR-3 whole cell lysate (20 µg).
Lane 6: Human SK-BR-3 nuclear lysate (20 µg).
Lane 7: Human Hs 578T whole cell lysate (20 µg).
Lane 8: Human Hs 578T nuclear lysate (20 µg).

Predicted band size: 59 kDa

Key control points

In the experiment, special attention should be given to key control points in addition to routine issues:

Sample preparation:

1. Add a complex protease inhibitor to avoid degradation of the target protein.
2. Select a suitable lysis buffer to enrich more target proteins.
3. Sonicate the cells to enrich the target protein.
4. Keep the sample on ice throughout the sample preparation process.
5. Determine the protein concentration of the sample through Bradford analysis, Lowry analysis, or BCA analysis.

Electrophoresis:

1. Load at least 20 μg total protein for electrophoresis.
2. We recommend using positive and negative controls.

Blocking:

1. There is no blocking solution applicable to all systems, please choose a suitable blocking solution.

Antibody incubation:

1. In the WB experiment, please avoid dry film conditions.
2. Please choose the appropriate antibody working concentration according to the product manual.
3. We recommend using fresh antibodies and not reusing antibodies.

References

1. Yuet-Kin Leung , Paul Mak, Sazzad Hassan and Shuk-Mei Ho. Estrogen receptor (ER)-beta isoforms: A key to understanding ER-beta signaling. Proc. Natl. Acad. Sci. USA (2006) 103(35):13162–13167. doi: 10.1073/pnas.0605676103
2. Ana Božović, Vesna Mandušić, Lidija Todorović and Milena Krajnović.  Estrogen Receptor beta: The promising biomarker and potential target in metastases. Int J Mol Sci. (2021) 22(4):1656. doi: 10.3390/ijms22041656
3. Nishant Gandhi, Gokul M. Metabolic reprogramming in breast cancer and its therapeutic implications. Cells. (2019) 8(2):89. doi: 10.3390/cells8020089
4. Yoko Omoto, Hirotaka Iwase. Clinical significance of estrogen receptor beta in breast and prostate cancer from biological aspects. Cancer Sci. (2015) 106(4):337-343. doi: 10.1111/cas.12613

PMID: 16938840, 33562134, 30691108, 25611678