Phospho-ETS1 (S251) Antibody

What is ETS1 and why is its phosphorylation at S251 significant?

ETS1 is the prototype member of the ETS (E26 transformation-specific) family of transcription factors. It was originally identified based on homology to the v-Ets oncogene isolated from the E26 erythroblastosis virus . ETS1 plays crucial roles in regulating gene expression and cellular processes including:

• Cell differentiation and proliferation

• Immune cell development and function

• Angiogenesis

• Cancer progression and metastasis

Phosphorylation at S251 is one of several regulatory phosphorylation events that control ETS1 function. According to research data, phosphorylation at S251, along with S282 and S285, occurs in response to calcium signaling via CaMK2/CaMKII and decreases ETS1's affinity for DNA . This represents an important mechanism for regulating the transcriptional activity of ETS1 in various cellular contexts.

Validating antibody specificity is critical for ensuring reliable experimental results. For Phospho-ETS1 (S251) antibodies, consider these validation approaches:

• Phosphatase treatment control: Treat half of your sample with lambda phosphatase before immunoblotting - the signal should disappear in the treated sample.

• Blocking peptide competition: Pre-incubate the antibody with the phosphorylated peptide used as the immunogen (synthetic phospho-peptide derived from human ETS1 around S251) . The specific signal should be significantly reduced.

• Genetic knockdown/knockout: Compare signal between wild-type cells and those with ETS1 knockdown/knockout.

• Phosphorylation-inducing conditions: Compare samples under conditions known to induce or reduce S251 phosphorylation (e.g., calcium signaling modulators that affect CaMK2 activity).

• Site-directed mutagenesis: Express wild-type ETS1 versus an S251A mutant that cannot be phosphorylated at this position.

What sample preparation techniques best preserve ETS1 phosphorylation status?

Preserving phosphorylation status is critical when studying phospho-proteins like ETS1. Based on research practices, the following protocols are recommended:

• Rapid sample collection and processing: Minimize the time between sample collection and lysis/fixation to prevent phosphatase activity.

• Phosphatase inhibitors: Always include a comprehensive phosphatase inhibitor cocktail in lysis buffers. For ETS1, include inhibitors targeting serine/threonine phosphatases.

• Cold processing: Keep samples and buffers at 4°C during processing to minimize enzymatic activity.

• Appropriate lysis buffers: For Western blot analysis, use RIPA or NP-40 based buffers supplemented with phosphatase inhibitors.

• Fixation for IHC/IF: For tissue samples, rapid fixation with paraformaldehyde or other appropriate fixatives helps preserve phosphorylation status.

• Storage conditions: Store lysates at -80°C and avoid repeated freeze-thaw cycles, as mentioned in product documentation .

How do different experimental conditions affect ETS1 S251 phosphorylation?

ETS1 phosphorylation at S251 is dynamically regulated by various cellular conditions:

• Calcium signaling: Research shows that calcium signaling activates CaMK2/CaMKII, which phosphorylates ETS1 at S251, S282, and S285 . Consider using calcium ionophores (like ionomycin) or calcium chelators (like BAPTA-AM) to modulate this pathway.

• Cell activation status: In T helper cells, ETS1 phosphorylation events occur during cell activation , suggesting that cellular activation status influences phosphorylation patterns.

• Cell type differences: Expression patterns of ETS1 vary between tissues, with high expression in lymphoid cells but low or absent expression in brain and kidney tissues , potentially affecting phosphorylation patterns.

• Disease states: In clear cell renal cell carcinoma (ccRCC), ETS1 expression is upregulated compared to normal tissues , which may be accompanied by altered phosphorylation patterns.

When designing experiments, these factors should be considered and appropriately controlled.

How can I study the relationship between ETS1 S251 phosphorylation and DNA binding activity?

To investigate how S251 phosphorylation affects ETS1's DNA binding capacity, consider these methodological approaches:

• Electrophoretic Mobility Shift Assay (EMSA): Compare DNA binding activity of phosphorylated versus non-phosphorylated ETS1. Research has shown that phosphorylation at S251, along with S282 and S285, decreases affinity for DNA .

• Chromatin Immunoprecipitation (ChIP): Use both total ETS1 antibody and Phospho-ETS1 (S251) antibody to compare occupancy at known ETS1 target sites under different conditions.

• In vitro DNA binding assays: Use recombinant ETS1 proteins (wild-type and S251 phospho-mimetic mutants) in surface plasmon resonance or fluorescence anisotropy assays to measure binding kinetics.

• ETS1 Transcription Factor Activity Assay: Commercial kits like the one described in search result can measure ETS1 activity, which could be correlated with phosphorylation status.

• Mutations studies: Compare DNA binding of wild-type ETS1 with phospho-mimetic (S251D/E) and non-phosphorylatable (S251A) mutants.

What is the interplay between different phosphorylation sites on ETS1?

ETS1 contains multiple phosphorylation sites that create a complex regulatory code. Based on the search results:

• Pointed domain phosphorylation: ERK2 phosphorylates ETS1 at T38 and S41, which enhances transcriptional activation by stimulating CBP recruitment .

• Central phosphorylation at S251, S282, S285: CaMK2/CaMKII phosphorylates these sites in response to calcium signaling, decreasing DNA binding affinity .

• Tyrosine phosphorylation at Y283: Src family kinases phosphorylate this site, which prevents COP1 (an ubiquitin ligase component) from binding and targeting ETS1 for degradation .

Research by Grenningloh et al. indicates that "both activating and inhibitory phosphorylation events of Ets-1 occur simultaneously and independently of each other during Th cell activation" . This suggests a complex regulatory system where different kinases act on ETS1 concurrently.

To study this interplay experimentally:

• Use phospho-specific antibodies against different sites (T38, S251, Y283) in parallel

• Employ kinase inhibitors to selectively block specific phosphorylation events

• Create multi-site phosphorylation mutants to assess combinatorial effects

What is the significance of ETS1 phosphorylation in cancer research?

ETS1 phosphorylation has important implications in cancer research, as demonstrated by several studies:

• Clear cell renal cell carcinoma (ccRCC): Research indicates that ETS1 is overexpressed in ccRCC compared to normal kidney tissues, and high ETS1 expression correlates with better prognosis . The study found that "high ETS1 expression levels were closely linked to early tumor stage and prolonged survival time" .

• Immune infiltration: There is a significant positive correlation between ETS1 expression and immune cell infiltration in ccRCC, including B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells .

• Tumor Mutation Burden (TMB): Interestingly, "TMB in the ETS1-high expression group was significantly less than that in the ETS1-low expression group" , suggesting a relationship between ETS1 expression and genomic stability.

For researchers investigating cancer:

• Consider examining the phosphorylation status of ETS1 at S251 in tumor samples versus normal tissues

• Correlate S251 phosphorylation with clinical parameters and patient outcomes

• Investigate whether S251 phosphorylation affects ETS1's interaction with the tumor microenvironment

How can I optimize Phospho-ETS1 (S251) antibody use in different applications?

Each application requires specific optimization strategies:

For Western Blot (1:500-1:1000 dilution) :

• Use freshly prepared lysates with phosphatase inhibitors

• Include positive controls (tissues/cells known to express phosphorylated ETS1)

• Block with BSA rather than milk (milk contains phosphatases)

• Consider using PVDF membranes which may retain phosphoproteins better than nitrocellulose

For Immunohistochemistry (1:50-1:200 dilution) :

• Test multiple antigen retrieval methods (citrate buffer vs. EDTA)

• Optimize fixation time (overfixation can mask phospho-epitopes)

• Use positive control tissues (lymphoid tissues with high ETS1 expression)

• Consider tyramide signal amplification for low-abundance phospho-proteins

For Immunofluorescence (1:50-1:200 dilution) :

• Fix cells quickly after treatment to preserve phosphorylation

• Test different permeabilization methods (Triton X-100 vs. methanol)

• Co-stain with total ETS1 antibody (different species) to assess phosphorylation ratio

What are the common challenges when detecting phosphorylated ETS1 and how can they be addressed?

Several technical challenges can arise when working with phospho-specific antibodies:

• Low signal intensity: Phosphorylation is often substoichiometric

  • Solution: Use signal amplification methods, increase antibody concentration, or enrich for phosphoproteins using phospho-enrichment kits

• High background: Non-specific binding can obscure specific signals

  • Solution: Optimize blocking conditions, increase washing stringency, and titrate antibody concentration

• Phospho-epitope masking: Protein interactions or other post-translational modifications may block antibody access

  • Solution: Test different denaturation/unfolding conditions during sample preparation

• Phosphatase activity: Endogenous phosphatases can reduce phosphorylation signals

  • Solution: Use multiple phosphatase inhibitors and keep samples cold throughout processing

• Antibody cross-reactivity: Some phospho-antibodies may recognize similar phospho-epitopes

  • Solution: Validate specificity using phosphatase treatment, competing peptides, and S251A mutants

How can I design experiments to study the functional consequences of ETS1 S251 phosphorylation?

To investigate the functional impact of S251 phosphorylation:

• Phosphorylation site mutants: Generate expression constructs with:

  • S251A (cannot be phosphorylated)

  • S251D/E (phosphomimetic)

  • Wild-type ETS1 (control)

• Rescue experiments:

  • Knock down endogenous ETS1 using siRNA targeting UTRs

  • Rescue with wild-type, S251A, or S251D/E mutants

  • Assess phenotypic differences (transcription, proliferation, etc.)

• Regulated phosphorylation:

  • Manipulate calcium signaling to alter CaMK2 activity and S251 phosphorylation

  • Use specific CaMK2 inhibitors to prevent phosphorylation

  • Monitor downstream effects on gene expression

• Integration with other phosphorylation sites:

  • Create combination mutants (e.g., T38E/S251A) to assess interplay between activating and inhibitory phosphorylation

  • Research has shown these can occur simultaneously and independently

• Cellular context:

  • Compare effects in different cell types, particularly lymphoid cells where ETS1 is highly expressed

  • Assess impact in normal versus disease states like cancer

What emerging technologies can enhance the study of ETS1 phosphorylation dynamics?

Several cutting-edge approaches could advance understanding of ETS1 phosphorylation:

• Mass spectrometry-based phosphoproteomics: Enables comprehensive mapping of all phosphorylation sites and their relative stoichiometry under different conditions.

• Phospho-specific biosensors: Genetically encoded FRET-based sensors could enable real-time monitoring of ETS1 phosphorylation in living cells.

• Single-molecule studies: Techniques like single-molecule FRET could reveal how phosphorylation alters ETS1 conformation and DNA interaction dynamics.

• CUT&RUN or CUT&Tag: These techniques provide higher resolution data than ChIP-seq for mapping phospho-ETS1 genome occupancy.

• Cryo-EM structural studies: Could reveal how S251 phosphorylation induces conformational changes that affect DNA binding.

• Multi-omics integration: Correlating phospho-ETS1 levels with transcriptome, epigenome, and proteome data could provide systems-level insights into its function.

What are the key unanswered questions regarding ETS1 S251 phosphorylation?

Despite progress in understanding ETS1 phosphorylation, several important questions remain:

• Kinase specificity: While CaMK2 has been implicated , are there other kinases that can phosphorylate S251 under different conditions?

• Phosphatase regulation: Which phosphatases dephosphorylate S251, and how are they regulated?

• Functional specificity: How does S251 phosphorylation specifically affect ETS1 target gene selection versus other phosphorylation events?

• Temporal dynamics: What is the lifetime of S251 phosphorylation in various cellular contexts?

• Therapeutic implications: Could modulating ETS1 S251 phosphorylation have therapeutic benefits in diseases where ETS1 plays a role?

• Interaction remodeling: Does S251 phosphorylation alter ETS1's protein-protein interaction network?

As noted in one study, "although these studies have provided a clearer understanding of ETS1 function, many unanswered questions remain regarding ETS1 structure, regulation, and biologic function" .