Phospho-RELA (T505) Antibody

What is the biological significance of RelA T505 phosphorylation?

RelA (p65) phosphorylation at threonine 505 acts as a negative regulator of NF-κB function. Unlike other phosphorylation events that positively regulate NF-κB activity, T505 phosphorylation suppresses its ability to induce diverse cellular processes including apoptosis, autophagy, proliferation, and migration . This phosphorylation occurs during S-phase of the cell cycle and in response to specific DNA-damaging agents like cisplatin, providing a mechanism of crosstalk between NF-κB signaling and DNA replication stress . In vivo studies have confirmed that RelA T505 phosphorylation provides an important physiological regulatory mechanism that antagonizes and limits aspects of RelA function associated with tumor-promoting activities .

How does RelA T505 phosphorylation differ from other RelA phosphorylation sites?

RelA T505 phosphorylation stands apart from other phosphorylation sites in several key aspects:

• It functions as a negative regulator of NF-κB activity, whereas most other sites (like S536) positively regulate NF-κB function

• It is specifically induced by cisplatin and other DNA replication stress inducers, but not by TNFα or other common NF-κB activators

• It is mediated by Chk1 kinase rather than IKK family kinases that phosphorylate many other RelA sites

• T505 phosphorylated RelA associates with HDAC1 corepressor complexes rather than CBP coactivator complexes found with S468 phosphorylated RelA

• Its phosphorylation peaks during S-phase of the cell cycle, whereas S536 phosphorylation is highest in G2 phase

What phenotypic changes are associated with disruption of RelA T505 phosphorylation?

Mutation of the RelA T505 residue to alanine (preventing phosphorylation) results in:

• Enhanced resistance to cisplatin and other DNA replication stress-inducing agents

• Increased cell proliferation and migration in cell culture models

• Aberrant hepatocyte proliferation following liver partial hepatectomy or damage in mouse models

• Earlier onset of hepatocellular carcinoma in the N-nitrosodiethylamine mouse model

• Reduced survival in the Eμ-Myc mouse model of B-cell lymphoma

• Resistance to CHK1 inhibitors in cancer models

• Significant remodeling of the actin cytoskeleton with cells appearing larger and displaying more intense actin staining

What are the optimal conditions for detecting phospho-RelA T505 in Western blots?

For optimal detection of phospho-RelA T505 in Western blots:

• Use fresh cell lysates prepared with phosphatase inhibitors to prevent dephosphorylation

• The recommended dilution range is 1:500-1:2000 for most commercially available antibodies

• Blocking should be performed with 5% BSA in TBST rather than milk (which contains phosphatases)

• Use positive controls such as cisplatin-treated cells, which show approximately 2-fold enhancement of T505 phosphorylation after 12 hours of treatment

• Consider enriching nuclear fractions since phospho-T505 RelA is primarily nuclear

• Include appropriate loading controls and total RelA antibody on parallel blots to normalize phospho-signal

How can I validate the specificity of a phospho-RelA T505 antibody?

Validating phospho-RelA T505 antibody specificity requires:

• Comparing signal in wild-type cells versus rela -/- cells reconstituted with T505A mutant RelA, which should show significantly reduced signal

• Performing peptide competition assays using phospho-T505 peptide versus non-phosphorylated peptide

• Treating samples with lambda phosphatase to confirm phosphorylation-dependent signal

• Testing induction with known stimuli (e.g., cisplatin) versus non-inducing stimuli (e.g., TNFα)

• Using phospho-ELISA with both phosphorylated and non-phosphorylated peptides covering the T505 region to demonstrate specificity

• For immunohistochemistry applications, blocking with the phospho-peptide should eliminate specific staining as demonstrated in human breast carcinoma samples

What are the key differences between various commercial phospho-RelA T505 antibodies?

While multiple vendors offer phospho-RelA T505 antibodies, they differ in several aspects:

• Host species (typically rabbit)

• Clonality (most are polyclonal though some monoclonal options exist)

• Validated applications (most support WB and ELISA, some support IHC)

• Cross-reactivity (most react with human, mouse, and rat species)

• Immunogen design (synthetic phosphopeptides around T505 position, but exact sequence may vary)

• Storage buffer composition (typically PBS with glycerol, BSA and sodium azide)

• Recommended dilutions for different applications

What cell types and experimental models are most appropriate for studying RelA T505 phosphorylation?

Based on published research, the following models are particularly suitable:

• MEF cells (both wild-type and rela -/- reconstituted with wild-type or T505 mutant RelA)

• Human U-2 OS osteosarcoma cells, which show inducible T505 phosphorylation after cisplatin treatment

• Mouse models of hepatocellular carcinoma, where T505A mutation shows clear phenotypes

• Eμ-Myc mouse model of B-cell lymphoma, which demonstrates the role of T505 in cancer progression

• Cell cycle synchronized cultures to study the S-phase specific phosphorylation

• Systems with active DNA replication stress, as T505 phosphorylation is particularly relevant in this context

How can I effectively study the functional impact of RelA T505 phosphorylation?

To study functional impacts of T505 phosphorylation:

• Generate T505A (phospho-deficient) and T505D (phospho-mimetic) mutants of RelA

• Reconstitute rela -/- cells with these mutants for comparative studies

• Measure functional outcomes including:

  • Apoptotic response to cisplatin (annexin staining, caspase 3 activation)

  • Cell proliferation rates (MTS assay, cell cycle analysis by FACS)

  • Migration capacity (wound-healing assay with mitomycin C to block proliferation)

  • Actin cytoskeleton organization (phalloidin staining)

  • Gene expression changes using qPCR or RNA-seq (focusing on genes like NOXA, Bcl-xL, WAVE3, α-actinin 4)

• Use ChIP assays to examine RelA binding to target gene promoters in T505A vs wild-type cells

• Perform ReChIP experiments to identify T505 phosphorylation-dependent cofactor recruitment (e.g., HDAC1)

What stimuli effectively induce RelA T505 phosphorylation in experimental settings?

Stimuli that effectively induce RelA T505 phosphorylation include:

• Cisplatin (shows approximately 2-fold enhancement after 12h treatment)

• p14 ARF tumor suppressor induction

• Cell synchronization in S-phase

• DNA replication stress inducers including:

  • Mitomycin C (DNA cross-linking agent)

  • Hydroxyurea (ribonucleotide reductase inhibitor)

• Notably, TNFα, UV radiation, and topoisomerase inhibitors like etoposide and SN38 do NOT significantly induce T505 phosphorylation

How should I interpret changes in RelA T505 phosphorylation in the context of cancer studies?

When interpreting changes in RelA T505 phosphorylation in cancer:

• Increased phosphorylation generally suggests activation of negative regulatory mechanisms that may suppress tumor-promoting NF-κB activities

• Reduced phosphorylation may indicate loss of this tumor-suppressive mechanism, potentially contributing to enhanced oncogenic NF-κB activity

• Context matters: in some cancers, RelA T505 phosphorylation correlates with resistance to specific chemotherapeutics

• Consider RelA T505 phosphorylation alongside other markers of DNA replication stress (γH2AX, phospho-RPA2)

• Changes should be interpreted within the larger context of ATR/CHK1 pathway activation, especially when considering response to CHK1 inhibitors

• In mouse models, loss of T505 phosphorylation (T505A mutation) accelerates cancer progression in both liver cancer and B-cell lymphoma models

What are common challenges in detecting RelA T505 phosphorylation and how can they be overcome?

Common challenges and solutions include:

How can phospho-RelA T505 data be correlated with gene expression changes?

To correlate phospho-RelA T505 with gene expression:

• Perform ChIP-seq with phospho-T505 specific antibodies to identify genome-wide binding sites

• Use ReChIP techniques to identify co-factor recruitment differences between phosphorylated and non-phosphorylated RelA

• Correlate binding with RNA-seq data to identify genes specifically regulated by T505-phosphorylated RelA

• Focus on key genes known to be regulated in a T505-dependent manner:

  • Anti-apoptotic genes (e.g., Bcl-xL) are often repressed by T505 phosphorylation

  • Pro-apoptotic genes (e.g., NOXA) are induced in a T505-dependent manner

  • Migration-associated genes (WAVE3, α-actinin 4, cortactin, N-Wasp, integrin-α5) show increased expression when T505 phosphorylation is prevented

• Validate findings with reporter assays using promoters of identified target genes in cells expressing wild-type versus T505A RelA

How does RelA T505 phosphorylation integrate with the DNA damage and replication stress response pathways?

RelA T505 phosphorylation represents a critical node connecting NF-κB signaling with DNA damage and replication stress responses:

• T505 is phosphorylated by Chk1, a key kinase activated during the DNA replication checkpoint

• This phosphorylation occurs primarily during S-phase when DNA replication is active

• It forms part of a complex regulatory network where different phosphorylated forms of RelA control cell cycle progression

• In S-phase, Akt (which normally activates IKK) becomes inactivated, while Chk1 becomes activated and phosphorylates RelA

• This phosphorylation influences RelA's interaction with p100/p52 (NF-κB2), which plays a role in regulating key cell cycle genes

• Mutation of RelA T505 disrupts the DNA replication stress response and leads to resistance to CHK1 inhibitors

• This suggests a feedback loop where CHK1 modulates NF-κB activity through T505 phosphorylation, which in turn affects sensitivity to CHK1 inhibition

What is the potential therapeutic relevance of targeting pathways involving RelA T505 phosphorylation?

The therapeutic relevance of RelA T505 phosphorylation includes:

• Predicting response to CHK1 inhibitors: tumors with defective T505 phosphorylation show resistance to these agents

• Combination therapies: DNA-damaging agents that induce T505 phosphorylation (like cisplatin) might synergize with agents that depend on intact T505 phosphorylation

• Targeting the Chk1-RelA axis: developing drugs that enhance T505 phosphorylation might help suppress tumor-promoting NF-κB activities

• Biomarker potential: T505 phosphorylation status could serve as a biomarker for predicting response to certain chemotherapeutics

• Personalized medicine approaches: tumors with mutations affecting the T505 pathway might require alternative treatment strategies

• Novel drug development: understanding the structural changes induced by T505 phosphorylation could lead to drugs that mimic these effects

How can multi-omics approaches enhance our understanding of RelA T505 phosphorylation networks?

Multi-omics approaches can provide comprehensive insights by:

• Integrating phosphoproteomics data to identify other proteins modified in response to conditions that induce T505 phosphorylation

• Combining ChIP-seq of phospho-T505 RelA with RNA-seq to create comprehensive maps of direct and indirect gene targets

• Using proteomics to identify interaction partners specific to phospho-T505 RelA versus unphosphorylated RelA

• Applying metabolomics to understand how T505 phosphorylation affects cellular metabolism

• Conducting single-cell analyses to determine cell-to-cell variability in T505 phosphorylation and its consequences

• Performing time-course studies to map the temporal dynamics of signaling networks after induction of T505 phosphorylation

• Using structural biology approaches to understand how T505 phosphorylation alters RelA conformation and protein-protein interactions

What recent discoveries have changed our understanding of RelA T505 phosphorylation?

Recent advances include:

• Identification of RelA T505 as a critical regulator of the DNA replication stress response in vivo

• Discovery that RelA T505A mutation confers resistance to CHK1 inhibitors, suggesting a feedback relationship

• Finding that T505 phosphorylation regulates the expression of migration-associated genes and cytoskeletal organization

• Demonstration that RelA T505 phosphorylation regulates autophagy, expanding its known cellular functions

• Identification of NOXA as a key T505-dependent effector in cisplatin-induced cell death

• Evidence that T505 phosphorylation influences RelA's interactions with chromatin modifiers like HDAC1

• Confirmation of the physiological significance of T505 phosphorylation in multiple mouse models of cancer

What methodological advances are improving the study of site-specific RelA phosphorylation?

Methodological advances include:

• Development of highly specific phospho-antibodies for different RelA modification sites

• CRISPR-Cas9 technology enabling precise genomic editing of endogenous RelA phosphosites

• Improved mass spectrometry approaches for quantitative phosphoproteomics

• Single-cell techniques to study heterogeneity in RelA phosphorylation

• Proximity labeling methods to identify phosphorylation-specific interaction partners

• Live-cell imaging with phospho-specific sensors to track dynamic changes in RelA modification

• Enhanced computational methods to predict functional consequences of specific phosphorylation events

• Development of nuclear isolation protocols that preserve phosphorylation status

What are the key outstanding questions regarding RelA T505 phosphorylation?

Critical unresolved questions include:

• What is the three-dimensional structural impact of T505 phosphorylation on RelA conformation?

• How does T505 phosphorylation affect RelA's interaction with other NF-κB family members?

• Are there tissue-specific differences in the regulation and consequences of T505 phosphorylation?

• How does T505 phosphorylation interact with other post-translational modifications of RelA?

• What is the clinical significance of T505 phosphorylation status in human cancers?

• Could targeting the T505 phosphorylation pathway represent a viable cancer therapy approach?

• What role does T505 phosphorylation play in non-cancer pathologies involving NF-κB?

• How is the T505 phosphorylation pathway affected by aging and cellular senescence?

• What are the epigenetic consequences of altered T505 phosphorylation?

• Do germline or somatic mutations affecting the T505 residue occur in human diseases?