Phospho-RELA (Ser311) Antibody

What is RELA (Ser311) phosphorylation and why is it important in NF-κB signaling?

RELA (also known as p65) is a key subunit of the transcription factor NF-κB. Serine 311 represents a critical phosphorylation site mediated by zeta protein kinase C (ζPKC). This post-translational modification plays an essential role in activating NF-κB transcriptional activity after the complex has translocated to the nucleus following IκB degradation.

In ζPKC-deficient cells, NF-κB is transcriptionally inactive and RELA phosphorylation in response to tumor necrosis factor alpha (TNF-α) is severely impaired . Studies using site-directed mutagenesis demonstrate that changing Ser311 to alanine (S311A) severely impairs RELA transcriptional activity, blocks its anti-apoptotic function, and abrogates the interaction with the co-activator CBP . Functionally, this phosphorylation event is required for recruitment of RNA polymerase II to NF-κB target gene promoters, such as the interleukin-6 (IL-6) promoter .

The importance of Ser311 phosphorylation is highlighted by experiments showing that cells stably expressing the RELA S311A mutant display dramatically inhibited responses to various agonists (TNF-α, IL-1, anti-lymphotoxin-β receptor agonistic antibody), indicating that phosphorylation of this residue is essential for κB-dependent transcription .

How does the Phospho-RELA (Ser311) Antibody function in detecting NF-κB activation?

Phospho-RELA (Ser311) antibodies are carefully engineered to recognize RELA only when it is phosphorylated at Serine 311. Product specifications indicate these antibodies detect endogenous levels of NF-κB p65 exclusively when phosphorylated at this specific residue . The antibodies are validated through several approaches:

• Specificity testing confirms they react with wild-type protein phosphorylated by ζPKC but not with unphosphorylated protein or S311A mutants .

• Competition experiments demonstrate that incubation with the phospho-peptide used to generate the antibody inhibits antibody binding, while the dephospho-peptide has no effect .

• Validation in multiple species (human, mouse, rat) across various applications including Western blotting, immunoprecipitation, immunofluorescence, immunohistochemistry, and ELISA .

For experimental applications, researchers can monitor NF-κB activation dynamics by stimulating cells with agonists like TNF-α for different time intervals, then either:

• Directly detecting phosphorylated RELA in whole cell lysates via Western blotting

• Isolating RELA via immunoprecipitation followed by detection with the phospho-specific antibody

• Visualizing subcellular localization using immunofluorescence or immunocytochemistry

What technical considerations are important for sample preparation when using Phospho-RELA (Ser311) Antibody?

Successful detection of phosphorylated RELA (Ser311) requires careful attention to sample preparation protocols that preserve the phosphorylation state. Based on validated experimental approaches, researchers should consider:

For Western blotting and immunoprecipitation:

• Include phosphatase inhibitors in all lysis and extraction buffers

• Process samples rapidly at cold temperatures to minimize dephosphorylation

• For optimal temporal resolution of phosphorylation events, collect samples at multiple time points after stimulation (typically 0-60 minutes for TNF-α stimulation)

• When using recombinant expression systems, consider tags (e.g., HA-tag) that facilitate immunoprecipitation without interfering with phosphorylation

For chromatin immunoprecipitation (ChIP):

• Fix cells in 1% formaldehyde for 10 minutes

• Sonicate under conditions optimized to generate DNA fragments with an average size of 1 kilobase

• Preclear samples with protein A agarose beads before immunoprecipitation

• Use specific elution conditions: 25 mM Tris-Cl (pH 7.5), 10 mM EDTA, 0.5% SDS at 60°C

Storage and handling:

• Commercial antibody preparations are typically supplied in PBS with 50% glycerol and 0.02% sodium azide at pH 7.4

• Store at -20°C and avoid repeated freeze-thaw cycles

• Working concentration is typically 1 mg/mL

These methodological considerations ensure optimal detection sensitivity and specificity when working with Phospho-RELA (Ser311) Antibody.

How does Ser311 phosphorylation mechanistically regulate RELA transcriptional activity?

Ser311 phosphorylation controls RELA transcriptional activity through several well-defined molecular mechanisms:

Recruitment of transcriptional co-activators:
Phosphorylation of Ser311 is essential for the interaction between RELA and the transcriptional co-activator CBP (CREB-binding protein). TNF-α stimulation promotes this interaction in cells expressing wild-type RELA, but the interaction is dramatically reduced in cells expressing the S311A mutant . This recruitment mechanism is critical because:

• It facilitates the assembly of the transcriptional machinery at NF-κB target gene promoters

• ChIP experiments demonstrate that both CBP and RNA polymerase II recruitment to the IL-6 promoter are severely abrogated in RELA S311A cells compared to controls

• The interaction of endogenous CBP with endogenous RELA is inhibited in ζPKC-/- cells but can be rescued when HA-ζPKC is reintroduced

Integration with DNA binding:
Interestingly, phosphorylation of Ser311 does not affect NF-κB nuclear translocation or DNA binding. Electrophoretic mobility shift assays (EMSAs) show that both wild-type RELA and the S311A mutant are capable of interacting with κB oligonucleotide probes . This indicates that Ser311 phosphorylation specifically regulates the transcriptional activation function of RELA rather than its ability to bind DNA.

Anti-apoptotic function:
RELA phosphorylation at Ser311 is crucial for its anti-apoptotic function. Cells stably overexpressing wild-type RELA are significantly protected from TNF-α-induced apoptosis compared to control cells. In contrast, cells expressing the RELA S311A mutant undergo apoptosis at levels comparable to control cells, indicating that mutation of Ser311 leads to the loss of RELA's ability to restrain cell death in response to activation of the TNF-α pathway .

These mechanisms collectively explain how Ser311 phosphorylation serves as a critical regulatory switch for RELA transcriptional activity and biological function.

What is the interplay between Ser311 phosphorylation and other post-translational modifications of RELA?

RELA undergoes multiple post-translational modifications that collectively regulate its function. Ser311 phosphorylation participates in a complex regulatory network with other modifications:

Relationship with adjacent Lys310 modifications:
Ser311 is adjacent to Lys310, which undergoes both methylation and acetylation:

• Methylation-phosphorylation switch: Under basal conditions, SETD6 monomethylates RELA at Lys310 (K310me1). This modification is recognized by the ankyrin repeats of G9a-like protein (GLP), which promotes a repressed chromatin state at NF-κB target genes. Phosphorylation of Ser311 prevents the interaction of GLP with K310me1, disrupting this repression .

• Structural basis: Crystal structure analysis reveals that Ser311 of RELA participates in polar interactions with SETD6. Phosphorylation at this position causes electrostatic repulsion from SETD6, preventing Lys310 methylation .

• Acetylation connection: Acetylation of Lys310 enhances NF-κB transcriptional activity. Phosphorylation of Ser311 may indirectly promote this modification by preventing methylation .

Coordination with other phosphorylation sites:
RELA contains multiple phosphorylation sites including Ser276, Ser529, and Ser536:

• Ser276 phosphorylation: This residue is targeted by PKA or MSK1 in response to different stimuli. Interestingly, mutation of Ser311 does not affect phosphorylation of Ser276, and vice versa, suggesting these modifications can occur independently .

• Cooperative effects: Research indicates that phosphorylation of both Ser276 and Ser536 increases assembly of phospho-RELA with p300, which enhances acetylation of Lys310 .

This table summarizes the key interrelationships between RELA modifications:

This complex interplay creates a sophisticated regulatory code that fine-tunes NF-κB-dependent gene expression in response to diverse cellular signals .

How can Phospho-RELA (Ser311) Antibody be optimally employed in chromatin immunoprecipitation studies?

Chromatin immunoprecipitation (ChIP) using Phospho-RELA (Ser311) Antibody provides powerful insights into the genomic binding patterns of phosphorylated RELA and its association with specific gene regulatory elements. Based on published methodologies, an optimized protocol includes:

Detailed ChIP Protocol:

• Cell fixation and preparation:

  • Fix cells (1-5 × 10⁶) in 1% formaldehyde for 10 minutes at room temperature

  • Quench with 125 mM glycine for 5 minutes

  • Wash twice with cold PBS

  • Resuspend in lysis buffer (1% SDS, 50 mM Tris, 10 mM EDTA)

• Chromatin shearing:

  • Sonicate samples to generate DNA fragments averaging 1 kilobase

  • Verify shearing efficiency by agarose gel electrophoresis

  • Centrifuge at 12,000 × g for 10 minutes to remove insoluble material

• Immunoprecipitation:

  • Preclear chromatin with protein A agarose beads

  • Immunoprecipitate overnight with anti-phospho-RELA(Ser311) antibody

  • Include appropriate controls: IgG negative control and total RELA antibody

  • Add protein A agarose beads and incubate for 4-6 hours

  • Wash extensively with increasing stringency buffers

• Elution and analysis:

  • Elute protein-DNA complexes (25 mM Tris-Cl, pH 7.5, 10 mM EDTA, 0.5% SDS) at 60°C

  • Reverse cross-links (65°C overnight with proteinase K)

  • Purify DNA using column-based methods

  • Analyze by qPCR, microarray, or next-generation sequencing

Scientific applications of Phospho-RELA (Ser311) ChIP:

• Promoter occupancy analysis: Determine whether phosphorylated RELA binds specific promoters (e.g., IL-6) in response to stimuli like TNF-α

• Co-factor recruitment studies: Perform sequential ChIP (re-ChIP) to examine co-localization of phosphorylated RELA with other transcription factors or co-activators like CBP

• Temporal dynamics: Conduct time-course experiments to track the kinetics of phosphorylated RELA binding and subsequent recruitment of transcriptional machinery

• Genome-wide binding profiles: Combine ChIP with next-generation sequencing (ChIP-seq) to map all genomic binding sites of phosphorylated RELA at Ser311

This approach has revealed that phosphorylation of Ser311 is required for efficient recruitment of both CBP and RNA polymerase II to the IL-6 promoter, providing mechanistic insight into how this modification regulates transcription .

What strategies can address the challenge of distinguishing between different phosphorylated forms of RELA?

RELA undergoes phosphorylation at multiple sites including Ser276, Ser311, Ser529, and Ser536, creating significant analytical challenges. To effectively distinguish between these phosphorylated forms, researchers can implement several complementary strategies:

Antibody validation and specificity testing:

• Confirm antibody specificity using phosphorylation-deficient mutants (e.g., S311A)

• Perform peptide competition assays using phospho- and non-phospho-peptides

• Validate across multiple applications (WB, IP, IF, ChIP) and sample types

Multi-antibody approach:

• Use a panel of site-specific phospho-antibodies in parallel

• Create a phosphorylation profile by comparing signals across different sites

• Include total RELA antibody to normalize for expression levels

Mass spectrometry-based methods:

• Employ phospho-enrichment techniques (TiO₂, IMAC) prior to MS analysis

• Use parallel reaction monitoring (PRM) or multiple reaction monitoring (MRM) for quantitative measurement of specific phosphorylation sites

• Analyze the stoichiometry of phosphorylation at different sites

Genetic approaches:

• Utilize phosphomimetic (S→D) and phosphorylation-deficient (S→A) mutants

• Rescue experiments in RELA-deficient cells with site-specific mutants

• CRISPR-Cas9 knock-in of tagged RELA versions for isolation of endogenous proteins

Temporal analysis:
Different phosphorylation sites show distinct temporal patterns following stimulation:

• Ser311 phosphorylation occurs rapidly after TNF-α stimulation

• Compare phosphorylation kinetics across sites using time-course experiments

Inhibitor studies:

• Use specific kinase inhibitors to block phosphorylation at individual sites

• For example, PKC inhibitors will preferentially affect Ser311 phosphorylation

• MAP kinase inhibitors may affect other phosphorylation sites

This systematic approach allows researchers to distinguish between different phosphorylated forms of RELA and understand their specific contributions to NF-κB signaling and transcriptional regulation .

How can Phospho-RELA (Ser311) Antibody be used to study the structural basis of RELA function?

Phospho-RELA (Ser311) Antibody provides a valuable tool for investigating the structural implications of this modification on RELA function through several sophisticated approaches:

Structural analysis of protein-protein interactions:

• Co-immunoprecipitation studies: Using Phospho-RELA (Ser311) Antibody for co-IP experiments reveals how this modification affects interaction with binding partners:

  • TNF-α stimulation promotes interaction of wild-type RELA with CBP, but this interaction is dramatically reduced with the S311A mutant

  • The antibody can pull down protein complexes specifically containing phosphorylated RELA for proteomic analysis

• Proximity ligation assays (PLA): This technique can visualize in situ protein interactions specifically involving phosphorylated RELA at Ser311.

Domain-specific functional analysis:

Ser311 is located in the linker region between the dimerization (Rel homology domain) and transactivation domains of RELA . Using the antibody in conjunction with truncation mutants can reveal:

• How phosphorylation affects the conformation and accessibility of these domains

• Whether interdomain communication is regulated by this modification

Structural basis for the "methyl-phospho switch":

The antibody can help elucidate the mechanism of the regulatory switch between adjacent residues:

• Crystallographic studies show that unphosphorylated Ser311 participates in polar interaction with the main chain carbonyl oxygen of Q226 and van der Waals contact with P228 of SETD6

• Phosphorylation of Ser311 causes repulsion from SETD6, preventing methylation of Lys310

• This creates a "methyl-phospho switch" between Lys310 and Ser311 that regulates NF-κB function

Biophysical characterization:

When combined with techniques like hydrogen-deuterium exchange mass spectrometry (HDX-MS), the antibody can help identify:

• Conformational changes induced by Ser311 phosphorylation

• Regions of RELA that become more exposed or protected upon phosphorylation

• Allosteric effects on distant parts of the protein structure

In silico modeling validation:

The antibody can provide experimental validation for computational models of RELA:

• Models connecting histone peptide-bound GLP with DNA-bound NF-κB

• Molecular dynamics simulations of how Ser311 phosphorylation affects RELA structure and dynamics

These approaches collectively provide comprehensive insights into how Ser311 phosphorylation influences RELA structure and function at the molecular level .

What are the technical considerations for using Phospho-RELA (Ser311) Antibody in different experimental systems?

Successfully employing Phospho-RELA (Ser311) Antibody across diverse experimental systems requires careful consideration of several technical factors:

Cell and tissue type considerations:

• Expression level variations:

  • ζPKC expression and activity vary across cell types, affecting basal and stimulus-induced Ser311 phosphorylation

  • Primary cells versus cell lines may show different phosphorylation dynamics

  • Verify antibody performance in your specific cell type before conducting extensive studies

• Species cross-reactivity:

  • Commercial antibodies have been validated for human, mouse, and rat samples

  • Some antibodies may also work with other species like pig, bovine, horse, sheep, and dog based on sequence conservation

  • Always validate new species applications with appropriate controls

Application-specific optimizations:

Stimulus and treatment variables:

• Stimulation protocols:

  • TNF-α concentration (10-100 ng/ml) and timing (peak phosphorylation occurs within 15-30 minutes)

  • Other stimuli (IL-1, LPS) may induce different phosphorylation kinetics

  • Include appropriate positive controls (TNF-α treated samples)

• Inhibitor studies:

  • PKC inhibitors can serve as negative controls

  • Phosphatase inhibitors are essential in all buffers

  • Proteasome inhibitors may enhance detection by preventing degradation

• Genetic manipulation considerations:

  • Use RELA S311A mutants as specificity controls

  • ζPKC-deficient cells serve as negative controls

  • Consider the impact of tags (HA, FLAG) on epitope accessibility

By carefully addressing these technical considerations, researchers can maximize the utility of Phospho-RELA (Ser311) Antibody across diverse experimental systems while ensuring reliable and reproducible results .

How can Phospho-RELA (Ser311) Antibody be used to study cross-talk between NF-κB and other signaling pathways?

Phospho-RELA (Ser311) Antibody provides a precise tool for investigating the complex interplay between NF-κB and other cellular signaling networks:

Kinase pathway interconnections:

ζPKC-mediated phosphorylation of RELA at Ser311 serves as a critical node connecting PKC signaling to NF-κB activation. Using the antibody, researchers can:

• Map upstream activators of ζPKC that ultimately influence RELA phosphorylation

• Determine how various stimuli that activate PKC isoforms affect NF-κB signaling through Ser311 phosphorylation

• Identify cross-inhibition or cross-activation between PKC and other kinase cascades that impact this modification

Integration with epigenetic signaling:

Phospho-RELA (Ser311) Antibody can reveal how NF-κB signaling interfaces with epigenetic regulation:

• The methyl-phospho switch between Lys310 methylation and Ser311 phosphorylation connects NF-κB to histone-modifying enzymes like GLP and SETD6

• This connection can be studied using sequential ChIP (re-ChIP) with antibodies against phosphorylated RELA and various histone modifications

• The antibody can track how chromatin-modifying drugs affect the phosphorylation status of nuclear RELA

Multi-omics experimental design:

Combining Phospho-RELA (Ser311) Antibody with multi-omics approaches creates powerful research paradigms:

• Phospho-proteomics integration:

  • Immunoprecipitate with Phospho-RELA (Ser311) Antibody followed by mass spectrometry

  • Identify proteins specifically interacting with phosphorylated RELA

  • Compare interaction profiles between different stimuli or cell types

• Genomics correlation:

  • ChIP-seq with Phospho-RELA (Ser311) Antibody to map genomic binding sites

  • Integrate with RNA-seq to correlate binding with transcriptional outcomes

  • Compare with other transcription factor binding patterns to identify cooperative or antagonistic relationships

• Pathway perturbation analysis:

  • Monitor Ser311 phosphorylation after inhibiting various signaling pathways

  • Use siRNA/CRISPR screens to identify novel regulators of this modification

  • Employ mathematical modeling to predict pathway interactions

Stimulus-specific signaling integration:

Different stimuli activate distinct signaling pathways that may converge on NF-κB. The antibody allows researchers to determine:

• How different inflammatory stimuli (TNF-α, IL-1, LPS) versus non-inflammatory signals affect Ser311 phosphorylation

• Whether growth factor signaling (EGF, PDGF) cross-talks with NF-κB through this modification

• How metabolic stress or cellular damage signals integrate with NF-κB activation via Ser311 phosphorylation

These approaches collectively enable researchers to use Phospho-RELA (Ser311) Antibody as a specific probe to dissect the complex cross-talk between NF-κB and diverse cellular signaling networks .

What recent advancements have occurred in understanding the role of RELA Ser311 phosphorylation in disease contexts?

While the search results don't provide comprehensive information about recent disease-specific advancements, we can synthesize available data to identify important research directions where Phospho-RELA (Ser311) Antibody would be valuable:

Inflammatory disorders:

Given the central role of NF-κB in inflammation, RELA Ser311 phosphorylation likely contributes to inflammatory disease mechanisms:

• The antibody can be used to assess phosphorylation status in patient-derived samples from inflammatory conditions like rheumatoid arthritis, inflammatory bowel disease, or psoriasis

• Relationships between aberrant ζPKC activity and excessive NF-κB activation in chronic inflammation can be explored

• Therapeutic targeting of this specific phosphorylation event might offer precision approaches to inflammatory disorders

Cancer biology:

RELA's anti-apoptotic function, which is regulated by Ser311 phosphorylation, has significant implications for cancer:

• Studies show that cells expressing RELA S311A mutants lose protection against TNF-α-induced apoptosis

• The antibody can assess whether increased Ser311 phosphorylation correlates with therapy resistance in cancer cells

• Targeting the ζPKC-RELA axis might sensitize resistant tumors to apoptosis-inducing therapies

Neurodegenerative diseases:

NF-κB plays complex roles in neuroinflammation and neurodegeneration:

• The antibody could track neuroinflammatory responses through RELA phosphorylation in models of Alzheimer's, Parkinson's, or ALS

• Cell-type specific patterns of RELA Ser311 phosphorylation (neurons vs. glia) might reveal differential regulation in CNS

Metabolic disorders:

NF-κB signaling interfaces with metabolic regulation:

• Phospho-RELA (Ser311) Antibody could investigate how metabolic stress affects NF-κB activation through this specific modification

• Connections between obesity-related inflammation and RELA phosphorylation patterns could be explored

Methodological advances:

Recent technical developments enhance the utility of Phospho-RELA (Ser311) Antibody:

• Multiplexed detection systems allow simultaneous analysis of multiple phosphorylation sites

• Single-cell western blotting techniques can reveal cell-to-cell variation in phosphorylation patterns

• Advances in imaging mass cytometry enable spatial mapping of phosphorylated RELA in complex tissues

Therapeutic implications:

The critical role of Ser311 phosphorylation in RELA function suggests potential therapeutic approaches:

• Small molecule inhibitors targeting the ζPKC-RELA interaction

• Peptide mimetics that compete for binding at the Ser311 region

• Structure-guided drug design based on the SETD6-RelA peptide complex

Research utilizing Phospho-RELA (Ser311) Antibody in these contexts would significantly advance our understanding of NF-κB regulation in disease and potentially identify novel therapeutic strategies .