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

Estrogen Receptor alpha Target Introduction

Protein Function

• Estrogen Receptor alpha (ERα) is a member of the nuclear hormone receptor family NR3 subfamily, involved in target cell proliferation, differentiation, and homeostasis.
• As a classical nuclear receptor, ERα can regulate the transcription of target genes through estrogen response elements (EREs) and by binding to other transcription factors such as AP-1/c-Jun, c-Fos, ATF-2, etc.
• The membrane component of ERα can act as a membrane receptor and, together with the G protein-coupled receptor family, exert transcriptional regulation through the second messenger system.
• ERα is also an effective non-classical RNA-binding protein that regulates the post-transcriptional expression of stress response genes.
• Abnormal signaling pathways of ERα are associated with the occurrence and development of various tumors, such as breast cancer, ovarian cancer, prostate cancer, colon cancer, etc.

Protein Expression

• ERα is highly expressed in the uterus, followed by the ovaries, and lowly expressed in related tissues such as the heart, liver, kidneys, brain, and lungs. The expression of ERα is particularly low in male or young female animal tissues.
• ERα46 (isoform 3, molecular weight 46 kDa) is expressed not only in human osteoblasts, macrophages, and vascular endothelial cells but also in colon cancer cells, breast cancer cells, and other cells.
• When the positive control MCF-7 cells reach the contact inhibition state, ERα46 is highly expressed, exceeding ERα66 (isoform 1, molecular weight 66 kDa).

Protein Localization

• Cell nucleus, cytoplasmic membrane, cell membrane. A small portion is localized between the cell nucleus and the inner membrane.
• ERα is a nucleocytoplasmic shuttle protein that rapidly shuttles between the cell nucleus and the cytoplasm.

Image 2: ICC experimental result of Estrogen Receptor alpha protein, using Anti-Estrogen Receptor alpha antibody (ab16660). Green: Estrogen Receptor alpha, Red: alpha Tubulin, Blue: DAPI.

Isoforms & post-translational modifications

• Human (P03372): Isoform 1: 66 kDa (predicted); Isoform 2: 53 kDa (predicted); Isoform 3: 46 kDa (predicted); Isoform 4: 36 kDa (predicted)
• Mouse (P19785): 67 kDa (predicted)
• Rat (P06211): 67 kDa (predicted)
• Phosphorylation
• Glycosylation
• Methylation
• Palmitoylation

WB experiment tips

Precautions

• Please note that there may be differences in the expression levels of target proteins in different samples, and it is necessary to confirm the expression levels of target proteins before testing. We recommend setting positive and negative controls.
• The detection of ERα46 in WB experiments may be related to the type and growth status of the cells being tested. Therefore, it is necessary to ensure that the cell type is appropriate, and the status is normal before sample preparation.
• The phosphorylation modification of ERα may require conditional induction (such as 100 ng/ml EGF treatment for 5 minutes in MCF-7 cells), so conditional induction can help solve the problem of weak or no signal.

Positive control

• ERα: MCF-7 whole cell lysate
  Human uterine tissue lysate
  Rat pituitary tissue lysate
  Mouse brain tissue lysate

Negative control (no expression/weak expression)

• ERα: MDA-MB231 whole cell lysate
  Human ovarian tissue lysate
  Rat brain tissue lysate
  Mouse kidney tissue lysate

Figure 3: Western blot experiment results of Estrogen Receptor alpha protein, using Anti-Estrogen Receptor alpha (EPR4097) antibody (ab108398).

Lane 1: MCF-7 whole cell lysate.
Lane 2: T47-D whole cell lysate.

Predicted band size: 66 kDa
Detected band size: 67 kDa

Figure 4: WB experiment results of Estrogen Receptor alpha protein, Anti-Estrogen Receptor alpha (E15) antibody (ab32063).

Lane 1: Human uterus tissue lysate.
Lane 2: Human kidney tissue lysate.
Lane 3: Human brain tissue lysate.
Lane 4: Mouse uterus tissue lysate.
Lane 5: Mouse ovary tissue lysate.
Lane 6: Mouse kidney tissue lysate.
Lane 7: Mouse brain tissue lysate.
Lane 8: Rat uterus tissue lysate.
Lane 9: Rat ovary tissue lysate.

Predicted band size: 67 kDa
Predicted band size: 67 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 sufficient amount of composite protease inhibitor to avoid degradation of the target protein.
2. Keep the sample on ice throughout the sample preparation process.
3. Determine the total protein concentration of the sample through Bradford analysis, Lowry analysis, or BCA analysis.

Electrophoresis:

1. Load at least 20 μg total protein for electrophoresis.

Blocking:

1. Use a 5% skim milk or BSA solution to block the membrane at room temperature for 1 hour or overnight at 4°C.

References

1. Jean-Francois Arnal, Françoise Lenfant, Raphaël Metivier et al. Membrane and nuclear estrogen receptor alpha actions: From tissue specificity to medical implications. Physio Rev. (2017). 97(3): 1045-1087.
   doi: 10.1152/physrev.00024.2016.
2. Yichen Xu, Peiwei Huangyang, Ying Wang et al. ERα is an RNA-binding protein sustaining tumor cell survival and drug resistance. Cell. (2021).184(20):5215-5229.
   doi: 10.1016/j.cell.2021.08.036.
3. Muralidharan Anbalagan, Brian G Rowan. Estrogen receptor alpha phosphorylation and its functional impact in human breast cancer. Mol Cell Endocrinol. (2015). 418(3):264-272.
   doi: 10.1016/j.mce.2015.01.016.
4. Man Shiow Jiang , Gerald Warren Hart. A subpopulation of estrogen receptors are modified by O-linked N-acetylglucosamine. J Biol Chem. (1997).272(4):2421-2428.
   doi: 10.1074/jbc.272.4.2421.
5. Muriel Le Romancer , Isabelle Treilleux, Nicolas Leconte et al. Regulation of estrogen rapid signaling through arginine methylation by PRMT1. Mol Cell. (2008). 31(2):212-221.
   doi: 10.1016/j.molcel.2008.05.025.
6. Ali Pedram, Mahnaz Razandi, Robert J Deschenes et al. DHHC-7 and -21 are palmitoylacyltransferases for sex steroid receptors. Mol Biol Cell. (2012).23(1):188-199.
   doi: 10.1091/mbc.E11-07-0638.
7. Gilles Flouriot, Heike Brand, Stefanie Denger et al. Identification of a new isoform of the human estrogen receptor-alpha (hER-alpha) that is encoded by distinct transcripts and that is able to repress hER-alpha activation function 1. The EMBO Journal. (2000). 19: 4688-4700.
   doi: 10.1093/emboj/19.17.4688.
8. Tetsuji Moriyama, Yoshihiro Yoneda, Masahiro Oka et al. Transportin-2 plays a critical role in nucleocytoplasmic shuttling of oestrogen receptor-α. Scientific Reports. (2020). 10:18640.
   doi: 10.1038/s41598-020-75631-3.
9. Graziella Penot, Christine Le Péron, Yohann Mérot et al. The Human Estrogen Receptor-α Isoform hERα46 Antagonizes the Proliferative Influence of hERα66 in MCF7 Breast Cancer Cells. Endocrinology. (2005). 146(12):5474-5484. doi: 10.1210/en.2005-0866.