Phospho-ESR1 (Ser118) Antibody

What is the biological significance of ESR1 phosphorylation at Serine 118?

ESR1 (Estrogen Receptor alpha) phosphorylation at Serine 118 plays a critical role in regulating the receptor's transcriptional activity. This specific post-translational modification occurs within the activation function-1 (AF-1) domain of the receptor. Research indicates that S118 phosphorylation significantly impacts ESR1 function, as mutation of this site substantially inhibits ESR1 activity. Mechanistically, phosphorylation at S118 directs gene-specific recruitment of ERα and various transcriptional coregulators to ERα target gene promoters . This phosphorylation event can be catalyzed by multiple kinases, including ERK1/2 mitogen-activated protein kinases (MAPK) and the cyclin-dependent protein kinase Cdk7 , suggesting integration of multiple signaling pathways in the regulation of ESR1 function.

Which kinases are responsible for phosphorylating ESR1 at Serine 118?

The phosphorylation of ESR1 at Serine 118 is mediated by several kinases as indicated in the research data. Primary kinases documented to phosphorylate this site include:

• ERK1/2 mitogen-activated protein kinases (MAPK)

• Cyclin-dependent protein kinase Cdk7

• Cyclin A/CDK2

• Casein kinase 1 (CK1)

• LMTK3 (in vitro)

This multi-kinase regulation suggests that ESR1 S118 phosphorylation integrates signals from various cellular pathways, including cell cycle regulation, growth factor signaling, and other stimulus-dependent processes. The diversity of kinases involved indicates the importance of this phosphorylation site as a convergence point for multiple signaling networks regulating estrogen receptor function.

How does phosphorylation at S118 affect ESR1 function?

Phosphorylation at S118 has several documented effects on ESR1 function:

• Enhancement of transcriptional activity: Research indicates that phosphorylation at this site "probably enhances transcriptional activity" .

• Regulation of protein-protein interactions: Phosphorylation directs the recruitment of transcriptional coregulators including SRC-1, TIF-2, and AIB to ERα target gene promoters .

• Counterregulation mechanisms: Dephosphorylation at Ser-118 by protein phosphatase PPP5C specifically inhibits ESR1's transactivation activity .

• Self-regulation: Self-association of the receptor can induce phosphorylation .

The functional consequences of S118 phosphorylation demonstrate its central role in fine-tuning estrogen receptor signaling in response to various cellular conditions, making it a critical regulatory node in hormone-responsive tissues.

What types of Phospho-ESR1 (S118) antibodies are commercially available?

Based on the search results, there are multiple types of Phospho-ESR1 (S118) antibodies available for research use:

Each antibody type offers specific advantages depending on the research application, with polyclonal antibodies potentially recognizing multiple epitopes while monoclonal antibodies provide higher specificity for the phosphorylated S118 site. Researchers should select the appropriate antibody based on their specific experimental requirements and validation needs.

What are the optimal protocols for Western blot applications using Phospho-ESR1 (S118) antibody?

Based on the validated methodologies from the search results, the following protocol guidelines are recommended for Western blot applications using Phospho-ESR1 (S118) antibody:

• Sample preparation:

  • Use appropriately treated cells (e.g., MCF7 cells treated with estradiol to induce phosphorylation)

  • Prepare lysates using standard cell lysis buffers containing phosphatase inhibitors to preserve phosphorylation status

• Antibody dilution ranges:

  • Primary antibody: 1:500-1:2000 dilution is recommended

  • Secondary antibody: Follow manufacturer's recommendations for anti-rabbit detection systems

• Controls:

  • Positive control: Lysates from cells known to express phosphorylated ESR1 (e.g., estradiol-treated MCF7 cells)

  • Negative control: Include a phospho-blocking peptide control to confirm specificity

• Detection considerations:

  • The expected molecular weight for ESR1 is approximately 66 kDa

  • Phosphorylation may cause slight shifts in apparent molecular weight

For validation, one study showed specific detection of phosphorylated ESR1 in MCF7 cells treated with estradiol, with signal effectively blocked by phospho-peptide pre-treatment, confirming antibody specificity .

How should Phospho-ESR1 (S118) antibodies be validated for research applications?

Comprehensive validation of Phospho-ESR1 (S118) antibodies should include multiple approaches:

• Specificity validation:

  • Phospho-blocking peptide competition assays: Signal should be abolished when antibody is pre-incubated with the phosphopeptide corresponding to the S118 region

  • Comparison of signal between phosphorylated and non-phosphorylated samples

  • Testing in S118A mutant cells (where serine is replaced with alanine)

• Application-specific validation:

  • For Western blot: Demonstrate single band at expected molecular weight that disappears with phosphatase treatment

  • For IHC: Compare staining patterns with and without phospho-blocking peptide

  • For ChIP: Validate with known ERα target genes and compare with total ERα binding

• Treatment-dependent validation:

  • Demonstrate increased signal after treatments known to induce S118 phosphorylation (e.g., estradiol, growth factors)

  • Show decreased signal after treatment with kinase inhibitors that block S118 phosphorylation

Documentation of validation results should include images of control experiments and quantification of signal differences between experimental conditions to establish robust methodology.

What are the recommended storage and handling conditions for Phospho-ESR1 (S118) antibodies?

Based on manufacturer guidelines from the search results, the following storage and handling recommendations apply to Phospho-ESR1 (S118) antibodies:

• Long-term storage:

  • Store at -20°C for up to 1 year from the date of receipt

  • Antibodies are typically supplied in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide

• Short-term storage:

  • For frequent use, store at 4°C for up to one month

• Critical handling considerations:

  • Avoid repeated freeze-thaw cycles as this can degrade antibody quality

  • Aliquot antibody upon receipt if multiple uses are anticipated

  • Ensure proper centrifugation of antibody vial before opening

  • Follow manufacturer's recommendations for thawing frozen antibodies

• Working dilution preparation:

  • Prepare working dilutions fresh on the day of use

  • Dilute in appropriate buffer according to application (e.g., blocking buffer for Western blot)

Proper storage and handling are essential for maintaining antibody specificity and sensitivity, particularly for phospho-specific antibodies which can be more vulnerable to degradation.

How can Phospho-ESR1 (S118) antibodies be used in Chromatin Immunoprecipitation (ChIP) assays?

Chromatin Immunoprecipitation using Phospho-ESR1 (S118) antibodies allows researchers to identify genomic targets specifically bound by the phosphorylated form of the receptor. Based on the methodologies described in the search results, a recommended ChIP protocol would include:

• Sample preparation:

  • Cross-link protein-DNA complexes using formaldehyde (typically 1% for 10 minutes)

  • Isolate and sonicate chromatin to fragments of approximately 200-500 bp

  • Pre-clear chromatin using isotype-matched IgG and protein A-magnetic beads

• Immunoprecipitation:

  • Add phospho-ERα S118 specific antibody (1-5 μg) to pre-cleared chromatin

  • Include parallel IPs with pan-ERα antibody and IgG control

  • Add protein A-magnetic beads and incubate overnight at 4°C

  • Perform stringent washing to remove non-specific interactions

• Analysis approaches:

  • qPCR analysis of known ERα target promoters

  • ChIP-seq for genome-wide analysis of phospho-ERα binding sites

  • Compare binding profiles between phospho-ERα and total ERα

• Validation strategies:

  • Demonstrate estrogen-dependent recruitment

  • Show differential recruitment compared to total ERα

  • Confirm association with coregulators like SRC-1, TIF-2, and AIB

This approach enables researchers to distinguish between binding events mediated by total ERα versus those specifically involving the S118-phosphorylated form, providing insights into phosphorylation-dependent gene regulation.

What are the key differences between using polyclonal versus monoclonal phospho-ESR1 (S118) antibodies?

The choice between polyclonal and monoclonal phospho-ESR1 (S118) antibodies significantly impacts experimental outcomes:

When designing experiments, researchers should consider these differences and select the antibody type that best aligns with their specific research questions and technical requirements.

How does the phosphorylation status at S118 change in response to different stimuli and in different cell types?

The phosphorylation of ESR1 at S118 shows stimulus-dependent and cell type-specific regulation patterns:

Stimulus-dependent regulation:

• Estradiol (E2) treatment: Induces rapid phosphorylation at S118, as demonstrated in MCF7 cells

• Growth factor signaling: EGF, IGF-1, and other growth factors can induce S118 phosphorylation through MAPK pathway activation

• Cell cycle progression: Cyclin A/CDK2 phosphorylates S118 in a cell cycle-dependent manner

Cell type-specific considerations:

• Hormone-responsive cancer cells: Breast cancer cell lines like MCF7 show robust S118 phosphorylation in response to estradiol

• Hormone-independent contexts: Some cells may show constitutive S118 phosphorylation through growth factor pathway activation

• Normal versus cancer tissues: Differential regulation patterns may exist between normal breast tissue and breast carcinomas

Pathological significance:

• Breast cancer: Altered S118 phosphorylation patterns correlate with disease progression and treatment response

• Endocrine resistance: Increased S118 phosphorylation has been associated with tamoxifen resistance in some studies

Understanding these context-dependent phosphorylation patterns is crucial for interpreting experimental results and may have implications for therapeutic targeting in diseases where ESR1 signaling is dysregulated.

What is the relationship between ESR1 S118 phosphorylation and other post-translational modifications?

ESR1 undergoes multiple post-translational modifications that interact in complex ways with S118 phosphorylation:

• Interconnected phosphorylation events:

  • S118 phosphorylation occurs within a cluster of phosphorylation sites in the AF-1 domain

  • Other phosphorylation sites (S104, S106, S167) may work cooperatively or antagonistically with S118

• Relationship with ubiquitination:

  • ESR1 is subject to ubiquitination regulated by multiple E3 ligases including LATS1 via DCAF1

  • Ubiquitination leads to ESR1 proteasomal degradation

  • Deubiquitination by OTUB1 counteracts this process

  • UBR5 specifically recognizes and binds ligand-bound ESR1 for ubiquitination

  • The relationship between S118 phosphorylation and ubiquitination rate remains an active area of research

• Glycosylation interactions:

  • ESR1 contains O-linked N-acetylglucosamine modifications

  • Potential crosstalk between glycosylation and phosphorylation at nearby sites may create complex regulatory patterns

• Functional consequences of PTM interplay:

  • Different combinations of PTMs likely create distinct "codes" that direct ESR1 to specific gene targets

  • PTM patterns may influence protein-protein interaction profiles and subcellular localization

Understanding this complex PTM network is essential for interpreting the specific roles of S118 phosphorylation in different cellular contexts and may inform more precise therapeutic targeting strategies.

What are common issues when using Phospho-ESR1 (S118) antibodies and how can they be resolved?

Researchers frequently encounter several challenges when working with phospho-specific antibodies targeting ESR1 at S118:

• Low or no signal issues:

  • Potential causes: Rapid dephosphorylation during sample preparation, insufficient phosphorylation induction, antibody degradation

  • Solutions: Include phosphatase inhibitors in all buffers, confirm stimulation conditions (e.g., estradiol treatment timing), verify antibody storage conditions

• High background or non-specific binding:

  • Potential causes: Insufficient blocking, cross-reactivity with similar phospho-epitopes, too high antibody concentration

  • Solutions: Optimize blocking conditions, perform phospho-peptide competition as control , adjust antibody dilution (try 1:500-1:2000 range for WB)

• Inconsistent results between experiments:

  • Potential causes: Variable phosphorylation levels, batch-to-batch antibody variation, inconsistent sample preparation

  • Solutions: Standardize cell treatment protocols, consider using recombinant monoclonal antibodies for greater consistency , implement rigorous positive controls

• Discrepancies between detection methods:

  • Potential causes: Different antibody performance in various applications, epitope accessibility differences

  • Solutions: Validate antibody specifically for each application, optimize protocols for individual techniques

• Tissue-specific detection challenges:

  • Potential causes: Epitope masking in certain fixation conditions, tissue-specific phosphatase activity

  • Solutions: Test different antigen retrieval methods for IHC, adjust fixation protocols, increase phosphatase inhibitor concentration

Creating a systematic troubleshooting workflow and maintaining detailed records of optimization attempts will help researchers overcome these common challenges.

How can I optimize immunohistochemistry protocols for Phospho-ESR1 (S118) detection in tissue samples?

Optimizing IHC protocols for phospho-specific antibodies requires careful attention to preserving phosphorylation status:

• Tissue preparation considerations:

  • Fix tissues promptly after collection to prevent phosphatase activity

  • Consider using phosphatase inhibitor-containing fixatives

  • Standardize fixation times (typically 24-48 hours in 10% neutral buffered formalin)

• Antigen retrieval optimization:

  • Test both heat-induced epitope retrieval (HIER) methods:

    • Citrate buffer (pH 6.0)

    • EDTA buffer (pH 9.0)

  • Optimize retrieval time (usually 10-20 minutes)

• Blocking and antibody conditions:

  • Use 1:100-1:300 dilution range for primary antibody incubation

  • Extend primary antibody incubation to overnight at 4°C

  • Include phospho-blocking peptide controls in parallel sections

• Signal amplification considerations:

  • Consider using polymer-based detection systems for enhanced sensitivity

  • Test tyramide signal amplification for tissues with low expression levels

• Validation approaches:

  • Compare staining with and without phospho-blocking peptide

  • Include known positive controls (e.g., breast cancer tissues with confirmed S118 phosphorylation)

  • Compare adjacent sections stained with total ERα antibody

• Expected staining patterns:

  • Primary nuclear localization

  • Intensity may vary between tumor regions

  • Heterogeneous expression within tissue is common

The optimal protocol will need to be determined empirically for each tissue type and fixation method, with careful documentation of all optimization steps.

How is Phospho-ESR1 (S118) being studied in relation to endocrine therapy resistance?

Phosphorylation of ESR1 at S118 has emerged as a potential biomarker and mechanism for endocrine therapy resistance:

• Clinical correlations:

  • Increased S118 phosphorylation has been observed in some tamoxifen-resistant tumors

  • The ratio of phospho-S118 to total ESR1 may correlate with clinical outcomes

  • Growth factor receptor overexpression (e.g., HER2) can drive S118 phosphorylation independent of estrogen

• Mechanistic insights:

  • Phosphorylation at S118 may allow ESR1 to function in a ligand-independent manner

  • S118 phosphorylation can influence the recruitment of different coregulator complexes , potentially altering the response to selective estrogen receptor modulators (SERMs)

  • Cross-talk between growth factor signaling and ESR1 phosphorylation may bypass the need for estrogen binding

• Therapeutic implications:

  • Combining endocrine therapies with kinase inhibitors that reduce S118 phosphorylation

  • Development of biomarker assays to predict therapy response based on phosphorylation status

  • Potential for novel therapeutic agents that specifically target phosphorylated ESR1

• Current research approaches:

  • Phosphoproteomics to map comprehensive ESR1 modification patterns in resistant versus sensitive tumors

  • Development of patient-derived xenograft models with varying S118 phosphorylation levels

  • Structure-based drug design targeting the conformation of phosphorylated ESR1

This rapidly evolving research area may lead to more personalized approaches to endocrine therapy based on ESR1 phosphorylation status.

How do genome-wide binding patterns differ between total ESR1 and Phospho-ESR1 (S118)?

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) studies have revealed important differences in the genomic binding patterns of total versus phosphorylated ESR1:

• Binding site selectivity:

  • Phospho-ESR1 (S118) appears to bind a subset of total ESR1 binding sites

  • Certain genomic regions show preferential enrichment for phospho-ESR1

  • Phosphorylation status may influence binding to non-canonical estrogen response elements

• Coregulator recruitment patterns:

  • Phospho-ESR1 (S118) directs the recruitment of specific transcriptional coregulators including SRC-1, TIF-2, and AIB to target gene promoters

  • The coregulator complexes associated with phospho-ESR1 may differ from those bound to non-phosphorylated receptor

• Chromatin environment associations:

  • Phospho-ESR1 binding may correlate with specific histone modifications

  • Pioneer factor requirements may differ between phospho and non-phospho ESR1

  • Chromatin accessibility patterns may influence phospho-ESR1 binding preference

• Temporal binding dynamics:

  • Phospho-ESR1 may show distinct kinetics of recruitment to target genes

  • Early versus late estrogen-responsive genes may show differential dependence on S118 phosphorylation

  • Stimulus-specific binding patterns may emerge depending on the kinase pathway activated

• Functional output differences:

  • Gene sets regulated by phospho-ESR1 may represent specific biological pathways

  • Phosphorylation-dependent binding correlates with specific transcriptional outcomes

  • Integration of multiple signaling inputs may occur at phospho-ESR1 binding sites

These genome-wide differences provide insight into how post-translational modifications create functional diversity in transcription factor activity and may explain context-specific effects of ESR1 signaling.