Phospho-ESR2 (Ser105) Antibody

What is Phospho-ESR2 (Ser105) Antibody and what does it detect?

Phospho-ESR2 (Ser105) Antibody specifically detects endogenous levels of Estrogen Receptor-beta (ERβ) only when phosphorylated at Serine 105. ERβ (also known as NR3A2) is a member of the steroid/thyroid hormone receptor superfamily of nuclear receptors with a molecular weight of approximately 55 kDa, though multiple isoforms ranging from 36-59 kDa have been described . The antibody recognizes this specific post-translational modification, allowing researchers to study the phosphorylation status of ERβ in various experimental and clinical settings. This antibody is crucial for investigating the functional significance of ERβ phosphorylation in estrogen signaling pathways and its role in various biological processes, including cancer development and progression .

What are the primary applications for Phospho-ESR2 (Ser105) Antibody?

Phospho-ESR2 (Ser105) Antibody has demonstrated utility across multiple experimental platforms:

For Western blot applications, typical protocols involve using 5-20% SDS-PAGE gels and loading approximately 30 μg of sample under reducing conditions . For immunohistochemistry, the antibody reveals nuclear uniform expression in tissue sections, which should be completely abolished when the antibody is preincubated with phosphatase, confirming phospho-specificity . Researchers should include appropriate positive controls (e.g., MCF-7 cells) and negative controls (e.g., ERβ-silenced cells or ER-negative BT-20 cells) in their experimental design .

How is the specificity of Phospho-ESR2 (Ser105) Antibody validated?

Proper validation of Phospho-ESR2 (Ser105) Antibody is critical for ensuring reliable research outcomes. Based on established protocols, validation should include:

• Phosphatase treatment: Signal should be abolished when the antibody is preincubated with phosphatase, confirming specificity for the phosphorylated form .

• siRNA knockdown: Experiments with ERβ-silenced cells (e.g., siRNA treatment in MCF-7 cells) should demonstrate elimination of the antibody signal .

• Cell line controls: The antibody should show positive signal in ER-positive cell lines (e.g., MCF-7) but not in ER-negative cell lines (e.g., BT-20) .

• Western blot analysis: The antibody should recognize a single specific protein band of the expected molecular weight (~59 kDa) in positive control samples .

• Stimulation experiments: Treatment with relevant ligands (e.g., 17β-estradiol, DPN) should show temporal changes in phosphorylation status that align with known signaling dynamics .

These validation approaches collectively establish the specificity and reliability of the antibody for detecting the phosphorylated form of ERβ at serine 105.

How does estrogen stimulation affect ERβ phosphorylation at serine 105?

Stimulation with 17β-estradiol (E2) induces a time-dependent change in ERβ phosphorylation at serine 105. Studies have demonstrated that treatment of MCF-7 cells with 10 nmol/L E2 increases expression of S105-ERβ within 30 minutes, with sustained phosphorylation for approximately 1 hour, before returning to control levels by 24 hours . This contrasts with S118-ERα phosphorylation, which is more rapidly induced within 15 minutes of E2 treatment .

Interestingly, the ERβ-selective agonist DPN produces a different phosphorylation profile, causing a gradual increase in S105-ERβ expression with maximal levels observed at 24 hours . These distinct temporal patterns suggest different mechanisms of activation by various ligands, which may contribute to the diverse biological effects of ERβ activation in different tissues and disease states.

The kinetics of ERβ phosphorylation provide important insights into the temporal regulation of estrogen receptor signaling and may have implications for understanding the effects of selective estrogen receptor modulators (SERMs) in clinical applications.

What is the relationship between ERβ phosphorylation at serine 105 and breast cancer outcomes?

ERβ phosphorylation at serine 105 has emerged as a potential biomarker in breast cancer research. Studies have investigated S105-ERβ expression in tissue microarrays comprising both tamoxifen-resistant (n=108) and tamoxifen-sensitive (n=351) breast cancer cases . While ERα phosphorylation status has been established as a correlate of patient outcomes, the clinical significance of ERβ phosphorylation is still being elucidated.

Research has demonstrated that S105-ERβ expression can be detected in breast cancer tissues and shows nuclear localization patterns that can be abolished by phosphatase treatment, confirming the specificity of the signal . The relationship between S105-ERβ expression and clinicopathological parameters has been analyzed, with comparable positivity observed in both tamoxifen-resistant and tamoxifen-sensitive cohorts .

This suggests that while phosphorylation at this site may not directly predict tamoxifen resistance, it may have other biological and clinical implications that warrant further investigation. Researchers studying this relationship should employ validated antibodies in tissue microarray analysis, correlate expression data with clinical outcomes, and perform multivariate analysis to account for potential confounding factors.

What signaling pathways regulate ERβ phosphorylation at serine 105?

Multiple signaling pathways have been implicated in the regulation of ERβ phosphorylation at serine 105:

• MAPK Pathway: Studies on murine ERβ have revealed that EGF and Ras enhance 17β-estradiol (E2)-induced transcriptional activity of ERβ via MAPK-directed phosphorylation . The human S105 site is equivalent to murine S106, which is a known MAPK phosphorylation site .

• Growth Factor Signaling: The cross-talk between growth factor receptors and ERβ is evidenced by EGF-induced phosphorylation of ERβ at S105. This suggests integration between growth factor and estrogen signaling networks .

• Estrogen Receptor Signaling: Classical estrogen receptor activation through ligand binding can lead to phosphorylation of S105, with different temporal dynamics depending on the specific ligand (e.g., E2 versus DPN) .

These phosphorylation events affect ERβ function by modulating its transcriptional activity and facilitating the recruitment of coactivators such as SRC-1 and CBP . Researchers investigating these pathways should employ specific pathway inhibitors, time-course experiments, and phosphomimetic or phospho-deficient mutants to fully understand the functional significance of S105 phosphorylation.

How can Phospho-ESR2 (Ser105) Antibody be used to study ERβ interactions with other proteins?

Phospho-ESR2 (Ser105) Antibody enables sophisticated studies of ERβ interaction networks through several methodological approaches:

• Co-immunoprecipitation (Co-IP): For studying protein-protein interactions, researchers can use the antibody to immunoprecipitate phosphorylated ERβ from whole cell extracts (WCE), cytoplasmic extracts (CE), or nuclear extracts (NE) . The protocol typically involves:

  • Preincubating 300 μg of extract with the antibody overnight at 4°C

  • Adding protein G agarose beads

  • Eluting proteins with Laemmli buffer

  • Analyzing by SDS-PAGE and Western blotting with antibodies against potential interacting partners

• Immunofluorescence Co-localization: This approach allows visualization of spatial relationships between phosphorylated ERβ and other proteins of interest. A protocol involving:

  • Fixing cells with cold methanol:acetone (1:1)

  • Blocking with 1% goat serum and 0.3% Triton X-100

  • Dual staining with anti-ERβ and antibodies against potential interacting proteins (e.g., EGFR)

  • Visualization using fluorescently-labeled secondary antibodies

• Mass Spectrometry of Protein Complexes: For comprehensive identification of ERβ-interacting proteins, researchers can immunoprecipitate FLAG-tagged ERβ, elute with 6M urea, and identify associated proteins by mass spectrometry .

These approaches have been successfully employed to investigate ligand-dependent differences in ERβ-interacting proteins in various cellular contexts, including lung adenocarcinoma cells .

How do murine and human ERβ phosphorylation sites compare?

Alignment of murine and human ERβ protein sequences reveals important conservation of phosphorylation sites with implications for translational research:

• Sequence Homology: Human ERβ shares 93.4% and 93.9% amino acid sequence identity with mouse and rat ERβ, respectively .

• Conserved Phosphorylation Sites: The S124 MAPK phosphorylation site in human ERβ is conserved in murine ERβ, and human S105 is equivalent to murine S106 .

• Functional Conservation: Studies on murine ERβ have shown that phosphorylation of S106 and S124 within the AF-1 domain enhances transcriptional activity through recruitment of co-activators SRC-1 and CBP . Similar mechanisms likely operate for human ERβ at the equivalent sites.

• Cross-species Reactivity: Many antibodies, including those against phospho-S105 ERβ, show cross-reactivity between human, mouse, and rat ERβ due to the high sequence conservation .

This conservation facilitates translational research, allowing findings from mouse models to inform human biology. Researchers should nonetheless validate antibody cross-reactivity and consider species-specific differences in signaling pathways when designing experiments.

What role does phosphorylation of ERβ at serine 105 play in protein interactions with membrane receptors?

Phosphorylation of ERβ at serine 105 appears to modulate its interactions with membrane receptors, particularly EGFR, highlighting the cross-talk between estrogen and growth factor signaling pathways. Studies using time-resolved fluorescence spectroscopy methods such as pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS) have provided insights into these dynamic interactions .

Research has demonstrated that EGF stimulation can influence heteromultimerization between receptors, suggesting that phosphorylation events, including those at S105 of ERβ, may mediate these interactions . The formation of receptor complexes is also influenced by membrane lipid composition, particularly phosphatidylinositol 4,5-bisphosphate (PIP₂) levels .

For researchers investigating these interactions, advanced methodological approaches include:

• Fluorescence fluctuation spectroscopy (FFS): These techniques circumvent the optical diffraction limit to examine membrane protein multimerization .

• Protein mobility analysis: Measuring diffusion coefficients (typically 0.24-0.33 μm²/s for membrane receptors) can provide insights into complex formation and receptor dynamics .

• Pharmacological manipulation: Using PLC inhibitors or activators to alter PIP₂ levels can help elucidate the role of membrane lipids in receptor interactions .

These receptor interactions may have significant implications for understanding how ERβ signaling interfaces with growth factor pathways in normal physiology and disease states.

What technical considerations should be addressed when using Phospho-ESR2 (Ser105) Antibody in colorimetric cell-based ELISA assays?

When implementing colorimetric cell-based ELISA assays with Phospho-ESR2 (Ser105) Antibody, researchers should consider several technical aspects to ensure reliable results:

• Assay Sensitivity: The Phospho-ESR2 (Ser105) Colorimetric Cell-Based ELISA provides qualitative detection with sensitivity requiring at least 5000 cells/well .

• Species Reactivity: The assay demonstrates reactivity across human, mouse, and rat samples, making it versatile for comparative studies .

• Lysate-Free Approach: The cell-based format eliminates the need for separate lysate preparation, offering a streamlined workflow for high-throughput applications .

• Dynamic Range: The assay maintains linearity over a range of >5000 cells, with detection at 450 nm .

• Normalization Strategy: To account for well-to-well variations in cell number, researchers should normalize phospho-specific signals to total protein content or cell number using appropriate controls .

• Treatment Conditions: When studying effects of various treatments or inhibitors, careful optimization of treatment duration and concentration is essential to capture the temporal dynamics of ERβ phosphorylation .

This methodology is particularly valuable for screening compounds that modulate ERβ phosphorylation and for investigating signaling pathway interactions in various cell types.