Phospho-NR4A1 (Ser351) Antibody

What is the specificity of Phospho-NR4A1 (Ser351) Antibody?

Phospho-NR4A1 (Ser351) Antibody specifically detects endogenous levels of Nuclear Receptor NR4A1 only when phosphorylated at serine 351. This specificity is achieved through a careful purification process where antibodies are produced by immunizing rabbits with synthetic phosphopeptide and KLH conjugates, then purified by affinity-chromatography using epitope-specific phosphopeptide. Importantly, non-phospho specific antibodies are removed through chromatography using non-phosphopeptides, ensuring high specificity for the phosphorylated form . The antibody recognizes the peptide sequence around the phosphorylation site of Serine351 (L-P-S(p)-K-P) derived from Human Nuclear Receptor NR4A1 .

What species reactivity does the Phospho-NR4A1 (Ser351) Antibody demonstrate?

The Phospho-NR4A1 (Ser351) Antibody shows cross-reactivity with human, mouse, and rat species, making it versatile for comparative studies across these mammalian models . This cross-species reactivity is based on sequence homology at the Ser351 phosphorylation site and surrounding amino acids. The conservation of this phosphorylation site across species suggests its functional importance in NR4A1 regulation and signaling pathways.

What are the recommended applications for Phospho-NR4A1 (Ser351) Antibody?

The antibody has been validated for multiple applications including immunohistochemistry (IHC) , Western Blot (WB), and enzyme-linked immunosorbent assay (ELISA) . For Western Blot applications, the recommended dilution range is 1:500-2000, while for peptide ELISA, a dilution range of 1:5000-20000 is suggested . Each application requires specific optimization considering the sample type, experimental conditions, and detection system used.

How should I design experiments to study NR4A1 phosphorylation in response to cellular stress?

When designing experiments to study NR4A1 phosphorylation in response to cellular stress, consider implementing a serum starvation and replenishment protocol, which has been shown to recapitulate replication stress and elicit NR4A1 induction with altered protein localization . Start with cells at baseline serum-replete culture conditions, then subject them to 24 hours of serum starvation followed by serum replenishment. Collect samples at multiple time points (e.g., baseline, during starvation, and at 30 min, 60 min, and later intervals after replenishment) to capture the dynamic changes in NR4A1 phosphorylation and localization. Research indicates that different cell lines may show variable timelines for immediate early gene (IEG) induction, with NR4A1 expression typically peaking around 60 minutes after serum replenishment .

What controls should be included when using Phospho-NR4A1 (Ser351) Antibody?

For rigorous experimental design with Phospho-NR4A1 (Ser351) Antibody, include the following controls:

• Positive control: Lysates from cells treated with agents known to induce Ser351 phosphorylation (e.g., mitogenic or stress stimuli, as NR4A1 is phosphorylated at Ser351 by RPS6KA1 and RPS6KA3 in response to these conditions)

• Negative control: Samples treated with phosphatase to remove phosphorylation

• Total NR4A1 antibody control: To normalize phospho-specific signal to total protein levels

• Knockdown/knockout validation: Where possible, include NR4A1 knockdown or knockout samples to confirm antibody specificity

• Competing peptide control: Pre-incubation of the antibody with the immunizing phosphopeptide should abolish specific signal

This comprehensive control strategy allows for accurate interpretation of phosphorylation-specific signals and helps distinguish between changes in phosphorylation versus changes in total protein levels.

How does phosphorylation at Ser351 affect NR4A1 subcellular localization and function?

Phosphorylation at Ser351 plays a critical role in regulating NR4A1 subcellular localization and function. Single-cell imaging studies using phospho-NR4A1 (Ser351) antibodies have confirmed that various cellular stresses can trigger changes in both nuclear and cytoplasmic localization of NR4A1 . Under normal conditions, NR4A1 predominantly exhibits nuclear localization where it binds to gene bodies and restrains baseline expression of immediate early genes (IEGs). Upon phosphorylation, particularly in response to stress stimuli, NR4A1 can undergo translocation, which affects its gene regulatory functions .

Research has shown that compounds like BEL (a pharmacological agent) can inhibit p38 phosphorylation and impede NR4A1 cytoplasmic localization, which appears to restore the inhibitory effect of NR4A1 on certain signaling pathways like TGF-β . This suggests that phosphorylation status directly impacts the protein's ability to shuttle between cellular compartments and interact with different molecular partners, ultimately affecting its transcriptional regulatory functions and role in processes like apoptosis.

How can ChIP-seq be optimized when using Phospho-NR4A1 (Ser351) Antibody to study chromatin binding patterns?

When utilizing Phospho-NR4A1 (Ser351) Antibody for Chromatin Immunoprecipitation sequencing (ChIP-seq), several optimization strategies can enhance experimental success:

• Crosslinking optimization: Start with standard 1% formaldehyde for 10 minutes, but optimize time (5-15 minutes) based on preliminary results, as NR4A1 binding patterns to gene bodies rather than promoters may require different crosslinking conditions.

• Sonication parameters: Aim for chromatin fragments of 200-500bp, with careful optimization of sonication cycles and amplitude based on your cell type.

• Antibody validation: Prior to full ChIP-seq, validate antibody specificity and efficiency using ChIP-qPCR on known NR4A1 target regions identified in previous studies, such as immediate early genes (IEGs) .

• Sequential ChIP consideration: For investigating the relationship between phosphorylated NR4A1 and RNA Polymerase II, which has been shown to have overlapping binding patterns at IEG gene bodies , consider sequential ChIP to detect co-occupancy.

• Data analysis pipeline: Implement peak calling algorithms suitable for detecting broad binding patterns across gene bodies rather than sharp peaks at transcription start sites, as research has shown NR4A1 predominantly binds to gene bodies under baseline conditions .

• Temporal sampling: Include multiple time points in your experimental design to capture the dynamic nature of NR4A1 chromatin binding, which has been shown to change significantly in response to serum stress conditions .

What are the recommended methods for quantifying changes in NR4A1 phosphorylation in response to experimental treatments?

For quantitative assessment of changes in NR4A1 phosphorylation in response to treatments, multiple complementary approaches are recommended:

• Western blot analysis: Use the Phospho-NR4A1 (Ser351) Antibody at the recommended dilution of 1:500-2000 . Always normalize phospho-NR4A1 signal to total NR4A1 protein levels using a separate total NR4A1 antibody. Consider using automated densitometry software for unbiased quantification.

• Quantitative immunofluorescence: This method allows for single-cell analysis of phosphorylation levels and subcellular localization simultaneously. Use high-content imaging systems for automated quantification across large numbers of cells.

• Flow cytometry: For suspension cells or adherent cells that have been appropriately detached, phospho-flow cytometry can provide quantitative data at the single-cell level with high statistical power.

• ELISA-based methods: Quantitative sandwich ELISA or AlphaLISA techniques can be developed for high-throughput screening of phosphorylation changes across multiple samples.

• Mass spectrometry: For absolute quantification and identification of multiple phosphorylation sites simultaneously, phospho-proteomics approaches using targeted mass spectrometry can be employed.

Each method has specific advantages and limitations regarding sensitivity, specificity, throughput, and the ability to detect spatial information, so the choice should be guided by your specific research question and available resources.

How does NR4A1 phosphorylation status relate to its role in regulating immediate early genes (IEGs)?

NR4A1 phosphorylation status appears to be intricately linked to its regulatory role in immediate early gene (IEG) expression. Research has demonstrated an inverse relationship between NR4A1 chromatin binding and IEG expression . Under baseline conditions, NR4A1 predominantly binds to gene bodies of IEGs and restrains their expression. ChIP-seq analysis has revealed that NR4A1 binding to IEG gene bodies is maximal under baseline conditions but begins to decline after 24 hours of serum starvation, reaching its lowest level approximately 1 hour after serum replenishment .

This decline in NR4A1 binding coincides precisely with the peak expression of IEGs such as FOS, FOSB, JUND, JUNB, and JUN, which are typically undetectable under baseline conditions but peak around 30 minutes after serum replenishment . The tight sequential timing and inverse relationship between NR4A1 chromatin binding and IEG expression suggest an inhibitory role for NR4A1 on IEG expression. Phosphorylation events, particularly at Ser351, likely play a regulatory role in this process by affecting NR4A1's ability to bind chromatin and interact with transcriptional machinery.

What is the relationship between NR4A1 phosphorylation, subcellular localization, and apoptotic signaling?

Phosphorylation events, including at Ser351, can regulate this translocation process. Under certain stress conditions, phosphorylation may trigger the release of NR4A1 from chromatin and facilitate its export from the nucleus. Single-cell imaging studies using phospho-NR4A1 (Ser351) antibodies have confirmed that various cellular stresses can lead to changes in both nuclear and cytoplasmic NR4A1 localization .

The phosphorylation-dependent localization of NR4A1 creates distinct functional pools of the protein with different roles in cell fate decisions. Nuclear NR4A1 generally functions in transcriptional regulation, while mitochondrial translocation is associated with pro-apoptotic functions. Understanding this relationship is particularly relevant in cancer research, where NR4A1 has demonstrated both tumor-suppressive and oncogenic properties depending on its localization and the cellular context.

How can I differentiate between the effects of Ser351 phosphorylation and other post-translational modifications of NR4A1?

Differentiating between the specific effects of Ser351 phosphorylation and other post-translational modifications (PTMs) of NR4A1 requires a multi-faceted experimental approach:

• Site-specific mutant studies: Generate Ser351 phospho-dead (S351A) and phospho-mimetic (S351D/E) mutants for comparison with wild-type NR4A1 in functional assays. These can be expressed in NR4A1 knockout or knockdown cells to eliminate background effects of endogenous protein.

• Multiple modification-specific antibodies: Use antibodies targeting different PTMs of NR4A1, such as acetylation, which has been shown to increase NR4A1 stability. NR4A1 is known to be acetylated by p300/CBP and deacetylated by HDAC1 .

• Mass spectrometry analysis: Employ phospho-proteomics and other PTM-focused mass spectrometry approaches to simultaneously identify and quantify multiple modifications on NR4A1 under various experimental conditions.

• Pharmacological inhibitors: Use specific inhibitors of kinases (RPS6KA1 and RPS6KA3 are known to phosphorylate NR4A1 at Ser351 ) or other modifying enzymes to dissect the contribution of each modification pathway.

• Sequential ChIP or Co-IP: Combine antibodies against different PTMs to determine if these modifications co-occur on the same NR4A1 molecules or represent distinct subpopulations.

• Temporal dynamics: Map the sequence of PTM events following stimulation by performing time-course experiments with antibodies against different modifications.

This comprehensive approach can help delineate the specific roles of Ser351 phosphorylation versus other PTMs in regulating NR4A1 function, localization, and protein-protein interactions.

What are common issues when using Phospho-NR4A1 (Ser351) Antibody in Western blotting, and how can they be resolved?

When working with Phospho-NR4A1 (Ser351) Antibody in Western blotting, researchers may encounter several common challenges:

• High background signal:

  • Solution: Optimize antibody dilution (start with 1:500-2000 as recommended )

  • Increase blocking time or concentration (5% BSA in TBST is often effective for phospho-antibodies)

  • Include phosphatase inhibitors in all steps of sample preparation

  • Consider using a more sensitive detection system with lower antibody concentrations

• Weak or absent signal:

  • Solution: Ensure proper sample preparation with phosphatase inhibitors

  • Validate that your experimental conditions actually induce Ser351 phosphorylation

  • Consider enriching phosphorylated proteins using phospho-protein enrichment kits

  • Decrease wash stringency or time

  • Optimize protein loading (50-100 μg total protein per lane is often needed for detection of phosphorylated proteins)

• Multiple bands or unexpected molecular weight:

  • Solution: NR4A1 has multiple isoforms and runs at approximately 64 kDa by SDS-PAGE

  • Include positive controls with known phosphorylation status

  • Consider using gradient gels for better resolution

  • Validate bands using competing peptide controls or NR4A1 knockdown samples

• Inconsistent results between experiments:

  • Solution: Standardize lysate preparation protocols

  • Prepare fresh buffers with phosphatase and protease inhibitors

  • Store antibody according to manufacturer recommendations (-20°C)

  • Consider aliquoting antibody to avoid freeze-thaw cycles

  • Use internal loading controls consistently across experiments

How can I optimize immunohistochemistry protocols for Phospho-NR4A1 (Ser351) Antibody?

Optimizing immunohistochemistry (IHC) protocols for Phospho-NR4A1 (Ser351) Antibody requires attention to several critical parameters:

• Tissue fixation and processing:

  • Fresh frozen sections often preserve phospho-epitopes better than formalin-fixed paraffin-embedded (FFPE) tissues

  • If using FFPE tissues, limit fixation time and optimize antigen retrieval methods

  • Consider using neutral-buffered formalin rather than acidic fixatives that may affect phospho-epitopes

• Antigen retrieval optimization:

  • Test multiple methods: heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 8.0-9.0)

  • Optimize retrieval time (typically 15-30 minutes)

  • Include phosphatase inhibitors in retrieval buffers

• Blocking and antibody dilution:

  • Use 3-5% BSA or specialized blocking reagents designed for phospho-epitopes

  • Test a range of antibody dilutions around the recommended dilution

  • Consider longer incubation times (overnight at 4°C) with more dilute antibody solutions

• Detection system selection:

  • Amplification systems like tyramide signal amplification (TSA) can enhance sensitivity for low-abundance phospho-proteins

  • Polymer-based detection systems often provide cleaner backgrounds than avidin-biotin systems

• Controls and validation:

  • Include tissue sections known to express phosphorylated NR4A1

  • Run parallel sections with competing phosphopeptide to confirm specificity

  • Consider dual staining with total NR4A1 antibody to assess proportion of phosphorylated protein

• Signal development and counterstaining:

  • Carefully monitor signal development to avoid overdevelopment

  • Choose counterstains that won't obscure nuclear or cytoplasmic signals depending on expected NR4A1 localization

How is Phospho-NR4A1 (Ser351) Antibody being used to study chromatin regulation and gene expression?

Phospho-NR4A1 (Ser351) Antibody has become an important tool in studying chromatin regulation and gene expression, particularly in understanding the unique role of NR4A1 in restraining immediate early gene (IEG) expression. Current research applications include:

• Genome-wide chromatin binding studies: ChIP-seq experiments have revealed that NR4A1 predominantly binds to gene bodies under baseline conditions, with a binding pattern distinct from other IEG proteins such as FOS and MYC, which typically localize to transcription start sites (TSS) . This unique binding pattern appears to be functionally important in gene regulation.

• Co-occupancy analysis with RNA Polymerase II: Research has demonstrated that NR4A1-bound IEGs display broad RNA Pol II residency at gene bodies and 3'-UTRs, with RNA Pol II pile-ups overlapping precisely with those of NR4A1 . This co-occupancy can be studied using sequential ChIP-seq or parallel ChIP-seq experiments with phospho-NR4A1 and RNA Pol II antibodies.

• Dynamic regulation of chromatin binding: Time-course experiments following serum starvation and replenishment have revealed the dynamic nature of NR4A1 chromatin binding, with maximal binding under baseline conditions followed by progressive decline during stress conditions . This temporal regulation appears to be inversely correlated with IEG expression.

• Integration with other genomic approaches: Combining phospho-NR4A1 ChIP-seq with techniques like ATAC-seq, RNA-seq, and Hi-C has allowed researchers to connect NR4A1 phosphorylation status to broader changes in chromatin accessibility, gene expression, and three-dimensional chromatin architecture.

Future research directions may include single-cell approaches to understand cell-to-cell variability in NR4A1 phosphorylation and chromatin binding, as well as studies investigating how NR4A1 phosphorylation affects recruitment of chromatin modifiers and remodelers.

What is the role of NR4A1 Ser351 phosphorylation in cancer and metastasis research?

NR4A1 Ser351 phosphorylation has emerged as a significant area of investigation in cancer and metastasis research, with several important implications:

• Metastatic progression: Studies tracing early steps of metastasis using patient-derived circulating tumor cells (CTCs) have identified increased chromatin accessibility across gene bodies and 3'-UTRs of multiple immediate early genes (IEGs), with NR4A1 playing a regulatory role . The phosphorylation status of NR4A1 at Ser351 appears to influence these processes.

• Genomic stability: Overexpression or deletion of NR4A1 have been shown to have opposing effects on genomic stability and tumorigenicity . Phosphorylation at Ser351 likely influences these functions by affecting NR4A1's interaction with chromatin and transcriptional machinery.

• Stress adaptation: NR4A1 phosphorylation in response to replication stress may represent a mechanism by which cancer cells adapt to stressful conditions during metastatic spread. This adaptation involves regulated release of NR4A1 from chromatin and subsequent IEG expression .

• Therapeutic implications: Understanding the role of NR4A1 phosphorylation in cancer cells may lead to novel therapeutic approaches. Compounds that modulate NR4A1 phosphorylation or its downstream effects could potentially interfere with cancer progression .

• Biomarker potential: Phosphorylated NR4A1 might serve as a biomarker for stress adaptation in cancer cells, potentially indicating more aggressive disease or resistance to certain therapies.

Future research directions may include developing small molecule modulators of NR4A1 phosphorylation, investigating combination therapies targeting both NR4A1 and its downstream effectors, and further elucidating the context-dependent roles of NR4A1 phosphorylation in different cancer types and stages.

What are the key considerations for researchers working with Phospho-NR4A1 (Ser351) Antibody?

Researchers working with Phospho-NR4A1 (Ser351) Antibody should consider several key factors to ensure successful experiments and reliable data interpretation:

• Antibody specificity and validation: Verify the antibody's specificity for phosphorylated Ser351 through appropriate controls, including competing peptide experiments and phosphatase treatments .

• Sample preparation protocols: Include phosphatase inhibitors in all buffers and maintain samples at cold temperatures to preserve phosphorylation status. Consider the dynamic nature of phosphorylation when designing experimental timelines.

• Application-specific optimization: Different applications (Western blot, IHC, ChIP-seq, etc.) require specific optimization of antibody concentration, incubation conditions, and detection methods .

• Biological context: Interpret results in the context of NR4A1's known functions in transcriptional regulation, immediate early gene expression, apoptotic signaling, and stress responses .

• Storage and handling: Store the antibody at -20°C and avoid repeated freeze-thaw cycles to maintain activity over the recommended shelf life of 1 year .