Phospho-RELA (Ser529) Antibody

What is Phospho-RELA/NF-κB p65 (Ser529) and why is it important in cell signaling research?

Phospho-RELA/NF-κB p65 (Ser529) represents the NF-κB p65 protein phosphorylated specifically at serine residue 529. This phosphorylation is critically important in cell signaling research as it enhances nuclear transcriptional activity of the NF-κB complex. NF-κB p65, also known as RELA or NFKB3, is one of five members of the nuclear factor κB (NF-κB)/Rel family that play vital roles in cell proliferation, immune response, survival, and apoptosis mechanisms . The phosphorylation at Ser529 specifically occurs via casein kinase II (CKII) and contributes to the regulation of gene expression following activation of the NF-κB pathway . This specific post-translational modification serves as an important molecular switch in inflammatory and immune responses, making it a valuable target for investigating signaling pathways in numerous pathological conditions.

How does Phospho-RELA (Ser529) differ from other phosphorylation sites on the NF-κB p65 protein?

While NF-κB p65 contains multiple phosphorylation sites including serines 276, 529, 536, and 471, the Ser529 site has distinctive characteristics and functions. The Ser529 phosphorylation is specifically catalyzed by casein kinase II (CKII), whereas other sites are targeted by different kinases . In contrast to other phosphorylation events, p65/RelA-Ser529 phosphorylation has been specifically linked to autophagic processes in certain cell types, particularly in astroglial cells where it plays a role in clasmatodendrosis (an irreversible astroglial degenerative change) . Research evidence indicates that this specific phosphorylation occurs following disassociation of NF-κB from IκB induced by cytokines like TNF-α . Unlike other phosphorylation sites that may primarily affect DNA binding or protein-protein interactions, Ser529 phosphorylation appears to have more specialized roles in certain cellular contexts, particularly in relation to autophagy activation and cellular degeneration responses .

What are the optimal protocols for detecting Phospho-RELA (Ser529) in Western blot experiments?

For optimal detection of Phospho-RELA (Ser529) in Western blot experiments, researchers should follow these methodological guidelines:

• Sample Preparation: For cell lysates, treatment with TNF-α (20 ng/mL) for 10 minutes has been shown to effectively induce Ser529 phosphorylation . For enhanced phosphorylation signals, consider co-treatment with phosphatase inhibitors such as Calyculin A (100 nM) .

• Protein Loading: Load approximately 15 μg of total protein per lane for sufficient detection sensitivity .

• Membrane Selection: PVDF membranes are recommended for optimal protein transfer and antibody binding .

• Antibody Dilution: Use primary antibody at 1:1000 dilution for Western blotting applications . For Mouse Anti-Human Phospho-RelA/NF kappa B p65 (S529) Monoclonal Antibody, 1 μg/mL has been validated .

• Detection Conditions: Use reducing conditions and appropriate immunoblot buffer (e.g., Immunoblot Buffer Group 1) .

Expected results include detection of a specific band at approximately 65 kDa, which represents the phosphorylated form of RelA/NF-κB p65 . The specificity of this signal can be confirmed using appropriate positive controls (TNF-α treated samples) and negative controls (untreated samples) .

How should Phospho-RELA (Ser529) antibodies be utilized in flow cytometry experiments?

For intracellular flow cytometry applications using Phospho-RELA (Ser529) antibodies, the following methodology is recommended:

• Cell Preparation: For peripheral blood mononuclear cells (PBMCs), stimulation with appropriate activators (such as TNF-α) is necessary to induce phosphorylation .

• Fixation and Permeabilization: Implement a true-Phosᵀᴹ permeabilization buffer protocol to maintain phospho-epitope integrity. Specifically:

  • Fix cells with 4% paraformaldehyde for 10-15 minutes at room temperature

  • Permeabilize with buffer containing detergent (such as Triton X-100) to allow antibody access to intracellular targets

• Antibody Concentration: For flow cytometric staining, use 5 μL of PE-conjugated anti-NF-κB p65 Phospho (Ser529) antibody per million cells in 100 μL staining volume, or 5 μL per 100 μL of whole blood .

• Instrument Settings: When using PE-conjugated antibodies, utilize blue laser (488 nm) or green/yellow-green laser (532/561 nm) excitation .

• Controls: Include both positive controls (stimulated cells) and negative controls (unstimulated cells) to accurately determine the shift in fluorescence intensity representing the phosphorylated form .

Titration of the antibody concentration is recommended for each specific application to determine optimal signal-to-noise ratio. This method allows for quantitative analysis of p65 phosphorylation at the single-cell level, enabling researchers to study heterogeneity in NF-κB activation within cell populations.

How can Phospho-RELA (Ser529) antibodies be utilized to investigate the role of NF-κB signaling in autophagy?

Recent research has established important connections between p65/RelA-Ser529 phosphorylation and autophagic processes, particularly in neurological contexts. To effectively investigate this relationship, researchers can implement the following experimental approach:

• Model Systems: Utilize models where clasmatodendrosis (autophagic astroglial death) can be induced, such as in rat hippocampus following status epilepticus (SE) .

• Co-localization Studies: Implement dual immunohistochemistry/immunofluorescence to assess:

  • Co-localization of Phospho-RELA (Ser529) with autophagy markers (LC3-II and Beclin-1)

  • Nuclear translocation and accumulation of phosphorylated p65

  • Relationship with lysosomal markers like LAMP-1

• Functional Validation: Employ TNF-α neutralization experiments (using sTNFp55R infusion) to demonstrate causality between TNF-α signaling, p65/RelA-Ser529 phosphorylation, and autophagic processes .

• Quantification Methods: Calculate the percentage of cells displaying nuclear p65/RelA-Ser529 phosphorylation in conjunction with autophagic markers under various experimental conditions .

This methodological approach provides mechanistic insights into how p65/RelA-Ser529 phosphorylation may act as a molecular switch for autophagy activation. In research examining clasmatodendrosis, approximately 51% of astrocytes showed this phenomenon in control conditions, which was reduced to 17% following TNF-α neutralization, demonstrating a clear functional relationship between these signaling events . These findings highlight the potential of targeting this specific phosphorylation event in neuroinflammatory and neurodegenerative disease contexts.

What are the validated experimental approaches for studying TNF-α-induced Phospho-RELA (Ser529) signaling in human cell lines?

To effectively study TNF-α-induced Phospho-RELA (Ser529) signaling in human cell lines, researchers should consider the following validated experimental approaches:

• Cell Line Selection: HeLa human cervical epithelial carcinoma cells have been extensively validated for studying this pathway . Other responsive human cell types include peripheral blood mononuclear cells and various cancer cell lines.

• Stimulation Parameters:

  • TNF-α concentration: 20 ng/mL represents the optimal concentration for inducing detectable p65 phosphorylation

  • Stimulation duration: 10 minutes has been established as sufficient for maximal phosphorylation response

  • Co-treatment considerations: Addition of 100 nM Calyculin A (a phosphatase inhibitor) can enhance detection of phosphorylation signals

• Detection Methods:

  Method	Sample Preparation	Detection Reagent	Expected Outcome
  Western blot	Cell lysates, PVDF membrane	Anti-Phospho-RELA (Ser529) antibody (1:1000)	Band at ~65 kDa
  Flow cytometry	Fixed/permeabilized cells	PE-conjugated anti-Phospho-RELA (Ser529)	Population shift in treated samples
  Immunocytochemistry	4% PFA fixation, Triton X-100 permeabilization	Various conjugated anti-Phospho-RELA (Ser529)	Nuclear translocation

• Downstream Analysis: To confirm functional relevance, assess nuclear translocation (via nuclear/cytoplasmic fractionation or imaging) and transcriptional activity (using reporter assays for NF-κB-responsive promoters) .

This experimental paradigm provides a robust platform for investigating TNF-α-induced p65/RelA-Ser529 phosphorylation and its subsequent effects on cellular processes including inflammatory responses, cell survival, and autophagy regulation.

How can researchers troubleshoot inconsistent results when detecting Phospho-RELA (Ser529) in different cellular contexts?

Inconsistent detection of Phospho-RELA (Ser529) across different cellular contexts may stem from several methodological factors. Researchers can implement the following troubleshooting strategies:

• Stimulation Optimization:

  • Verify TNF-α potency and activity with appropriate bioassays

  • Establish cell-type specific time-course experiments (5-60 minutes) as phosphorylation kinetics vary across cell types

  • Consider pre-treatment with phosphatase inhibitors (e.g., Calyculin A) to prevent rapid dephosphorylation

• Sample Handling:

  • Implement rapid sample processing to minimize dephosphorylation

  • Include phosphatase inhibitors in all lysis buffers

  • For adherent cells, consider direct lysis in the culture dish to prevent signaling changes during harvesting

• Antibody Validation:

  • Perform phospho-epitope blocking experiments with competing phosphopeptides

  • Include appropriate positive controls (e.g., HeLa cells treated with TNF-α) in each experiment

  • Consider testing multiple antibody clones targeting the same phosphorylation site

• Cell Type-Specific Considerations:

  • Assess baseline NF-κB activation state in your cell model

  • Verify expression levels of TNF receptors (particularly TNFp75 receptor) as their abundance influences signaling

  • For neuronal/glial cells, consider cell-specific optimizations as these cells may respond differently than epithelial or immune cells

• Technical Validation:

  • Implement orthogonal methods for detection (e.g., mass spectrometry-based phosphoproteomics)

  • Confirm specificity by using phospho-deficient mutants (S529A) as negative controls

These systematic approaches address the major sources of variability in phospho-specific detection and help ensure reproducible results across diverse experimental conditions and cellular contexts.

What are the critical considerations for preserving Phospho-RELA (Ser529) epitopes during sample preparation for immunocytochemistry?

Preserving phospho-epitopes during immunocytochemistry requires careful attention to multiple steps in the sample preparation process. For optimal detection of Phospho-RELA (Ser529), researchers should implement these critical considerations:

• Fixation Parameters:

  • Use 4% paraformaldehyde (PFA) fixation, which has been validated for preserving phospho-RELA (Ser529) epitopes

  • Limit fixation time to 10-15 minutes at room temperature to prevent epitope masking

  • Avoid methanol fixation, which can extract phospholipids and potentially alter phospho-epitope conformation

• Permeabilization Optimization:

  • Use Triton X-100 for permeabilization, which has been specifically validated for phospho-RELA (Ser529) detection

  • Titrate permeabilization agent concentration (typically 0.1-0.3%) to balance antibody accessibility with epitope preservation

  • Consider gentle permeabilization methods for particularly sensitive samples

• Blocking Strategy:

  • Include phosphatase inhibitors in blocking buffers to prevent enzymatic dephosphorylation during staining

  • Use BSA rather than non-fat dry milk for blocking, as milk contains phosphoproteins that may interfere with phospho-specific antibodies

  • Consider adding phosphatase inhibitors to all washing buffers

• Antibody Incubation Conditions:

  • Optimize antibody concentration through careful titration experiments

  • For PE-conjugated antibodies, protect samples from light exposure to prevent photobleaching

  • Consider longer incubation times at 4°C rather than shorter incubations at room temperature

• Signal Amplification Considerations:

  • For low abundance phospho-signals, implement tyramide signal amplification or similar techniques

  • When using amplification methods, include additional controls to verify specificity

These critical considerations address the unique challenges associated with phospho-epitope preservation and detection, ensuring optimal visualization of Phospho-RELA (Ser529) in diverse experimental contexts. Implementing these recommendations will enhance signal specificity and reproducibility in immunocytochemical applications.

How should researchers interpret variations in Phospho-RELA (Ser529) levels in the context of TNF-α signaling pathways?

Interpreting variations in Phospho-RELA (Ser529) levels requires careful consideration of multiple factors within TNF-α signaling pathways. Researchers should apply the following interpretative framework:

• Temporal Dynamics: Phosphorylation at Ser529 typically occurs rapidly following TNF-α stimulation, with detectable levels within 10 minutes . Variations in this temporal profile may indicate:

  • Altered receptor sensitivity or expression

  • Modified upstream signaling kinetics

  • Presence of negative regulatory mechanisms

• Subcellular Localization: The biological significance of p65/RelA-Ser529 phosphorylation depends on its localization:

  • Nuclear accumulation suggests active transcriptional regulation

  • Cytoplasmic retention despite phosphorylation may indicate additional regulatory mechanisms

  • In glial cells, nuclear p65/RelA-Ser529 phosphorylation associated with watery nuclear dissolution indicates progression toward clasmatodendrosis

• Cell Type-Specific Thresholds:

  • Different cell types display varying thresholds for phosphorylation-dependent responses

  • In astrocytes, approximately 51% exhibit clasmatodendrosis with nuclear p65/RelA-Ser529 phosphorylation under pathological conditions

  • Reductions in this percentage (to ~17% following TNF-α neutralization) represent significant biological effects

• Integration with Other NF-κB Modifications:

  • Consider Ser529 phosphorylation in relation to other post-translational modifications of p65

  • Multiple simultaneous modifications may create specific "barcode" patterns with distinct functional outcomes

  • The absence of other p65/RelA phosphorylation events (e.g., at Ser276, Ser536) alongside Ser529 phosphorylation may have specific biological implications

• Pathway Crosstalk Interpretation:

  • Purinergic receptor (P2X7) activation attenuates clasmatodendrosis and reduces NF-κB DNA-binding activity, suggesting pathway interactions that influence Ser529 phosphorylation

  • Consider TNF receptor subtype expression levels (particularly TNFp75) when interpreting response magnitudes

This interpretative framework provides a comprehensive approach to understanding variations in p65/RelA-Ser529 phosphorylation, moving beyond simple quantification to mechanistic insights regarding TNF-α signaling dynamics and biological consequences.

What are the implications of Phospho-RELA (Ser529) in neurodegenerative disease research?

Research on p65/RelA-Ser529 phosphorylation has revealed significant implications for neurodegenerative disease mechanisms and potential therapeutic interventions:

• Clasmatodendrosis Connection: p65/RelA-Ser529 phosphorylation has been specifically linked to clasmatodendrosis, an irreversible astroglial degenerative change characterized by extensive swelling, vacuolization, and beaded processes . This process appears to represent an autophagic form of astroglial death with particular relevance to:

  • Epilepsy models (status epilepticus)

  • Potential relevance to other neurodegenerative conditions with glial pathology

• Autophagy Regulation: The direct relationship between p65/RelA-Ser529 phosphorylation and autophagy markers (LC3-II, Beclin-1, LAMP-1) in glial cells provides new insights into:

  • Mechanisms of selective cellular vulnerability in neurodegeneration

  • Potential targets for modulating autophagy in glial cells

  • Novel approaches to neuroprotection through glial preservation

• TNF-α Pathway as Therapeutic Target: The demonstrated reduction in clasmatodendritic astrocytes following TNF-α neutralization (from 51% to 17%) suggests:

  • Potential therapeutic value of TNF-α inhibition in conditions with glial pathology

  • Importance of specifically targeting the p65/RelA-Ser529 phosphorylation process

  • Possible benefits of casein kinase II inhibition in preventing detrimental astroglial changes

• Biomarker Potential: Nuclear p65/RelA-Ser529 phosphorylation could serve as:

  • A marker for early astroglial stress before irreversible clasmatodendrosis

  • A potential diagnostic indicator of neuroinflammatory processes

  • A tool for monitoring therapeutic efficacy in interventional studies

• Cellular Interaction Mechanisms: The relationship between p65/RelA-Ser529 phosphorylation and TNFp75 receptor expression suggests:

  • Cell-specific vulnerability based on receptor expression patterns

  • Potential for targeted intervention based on receptor profiles

  • Importance of neuron-glia interactions in disease progression

These research implications highlight the unique value of studying p65/RelA-Ser529 phosphorylation in neurodegenerative contexts, offering mechanistic insights and potential therapeutic approaches for conditions involving pathological glial changes and neuroinflammation.

How might phospho-proteomics approaches enhance our understanding of Phospho-RELA (Ser529) in complex signaling networks?

Phospho-proteomics approaches offer significant potential to advance our understanding of Phospho-RELA (Ser529) within complex signaling networks through several methodological innovations:

• Multiplexed Phosphorylation Profiling: Modern phospho-proteomics can simultaneously quantify multiple phosphorylation events on p65/RelA (Ser276, Ser529, Ser536, Ser471) to determine:

  • Temporal hierarchy of phosphorylation events following TNF-α stimulation

  • Cell type-specific phosphorylation "barcodes" that may dictate distinct functional outcomes

  • Correlations between Ser529 phosphorylation and other post-translational modifications

• Kinase Activity Profiling: Phospho-proteomics can map casein kinase II (CKII) activity networks to:

  • Identify additional substrates co-regulated with p65/RelA-Ser529

  • Reveal compensatory phosphorylation events following CKII inhibition

  • Clarify the intersection between TNF-α signaling and CKII activation pathways

• Temporal Dynamics Analysis:

  • Pulse-chase phospho-proteomics can determine the precise half-life of Ser529 phosphorylation

  • Reveal the relationship between phosphorylation dynamics and functional outcomes like gene expression

  • Identify phosphatases responsible for Ser529 dephosphorylation in different cellular contexts

• Interactome Analysis:

  • Phospho-dependent interaction networks can be mapped using proximity labeling combined with phospho-enrichment

  • This approach would reveal how Ser529 phosphorylation alters p65/RelA protein-protein interactions

  • Identify specific transcriptional cofactors recruited in a Ser529 phosphorylation-dependent manner

• Single-Cell Applications:

  • Emerging single-cell phospho-proteomics could reveal cell-to-cell heterogeneity in p65/RelA-Ser529 phosphorylation

  • This would be particularly valuable in understanding why only subsets of astrocytes undergo clasmatodendrosis despite uniform TNF-α exposure

  • Could identify previously unrecognized cell states based on phosphorylation patterns

These phospho-proteomics approaches would transform our understanding of p65/RelA-Ser529 phosphorylation from a single binary event to a component of complex, multidimensional signaling networks with context-specific functions and regulatory mechanisms.

What research questions remain unanswered regarding the role of Phospho-RELA (Ser529) in disease pathogenesis beyond neurodegeneration?

Despite progress in understanding p65/RelA-Ser529 phosphorylation in neurodegeneration, several critical research questions remain unexplored in other disease contexts:

• Cancer Biology Questions:

  • How does p65/RelA-Ser529 phosphorylation contribute to tumor-specific NF-κB activation patterns?

  • Does selective targeting of this phosphorylation event offer advantages over broad NF-κB inhibition in cancer therapy?

  • What is the relationship between human papillomavirus 16 E7 and p65/RelA-Ser529 phosphorylation in oral cancer cells as suggested by preliminary research?

• Inflammatory Disease Mechanistic Questions:

  • Does p65/RelA-Ser529 phosphorylation regulate specific subsets of inflammatory genes distinct from other phosphorylation events?

  • How do chronic inflammatory conditions alter the dynamics and functional consequences of this phosphorylation?

  • Could targeting this specific phosphorylation offer selective anti-inflammatory effects with reduced side effects?

• Metabolic Regulation Questions:

  • What is the role of p65/RelA-Ser529 phosphorylation in metabolic inflammation?

  • How does this phosphorylation respond to metabolic stressors like hyperglycemia or hyperlipidemia?

  • Is there crosstalk between nutrient-sensing pathways and p65/RelA-Ser529 phosphorylation?

• Developmental Biology Questions:

  • What role does p65/RelA-Ser529 phosphorylation play in NF-κB-dependent developmental processes?

  • How is this phosphorylation regulated during cellular differentiation and tissue specialization?

  • Given NF-κB p65's crucial role in skeletal development through regulation of chondrocyte and osteoblast differentiation , what specific functions might Ser529 phosphorylation serve in these processes?

• Therapeutic Development Questions:

  • Can small molecules selectively inhibit p65/RelA-Ser529 phosphorylation without affecting other NF-κB functions?

  • Would targeting the casein kinase II-p65/RelA axis provide therapeutic benefits in specific disease contexts?

  • How might combination therapies targeting both TNF-α signaling and p65/RelA-Ser529 phosphorylation be optimized?

Addressing these research questions would significantly expand our understanding of p65/RelA-Ser529 phosphorylation beyond its established role in neurodegeneration, potentially revealing new therapeutic approaches for diverse pathological conditions linked to dysregulated NF-κB signaling.