Phospho-RELA (Thr435) Antibody

What is the biological significance of RelA (p65) Thr435 phosphorylation?

Thr435 phosphorylation provides a crucial regulatory mechanism for modulating NF-κB transcriptional activity in a gene-specific manner. Research has demonstrated that this post-translational modification affects the ability of RelA to interact with chromatin-modifying enzymes, particularly HDAC1 (histone deacetylase 1) . The phosphorylation state of Thr435 can significantly influence the expression of specific target genes rather than globally affecting all NF-κB-dependent genes. For example, the phosphomimetic T435D mutation has been shown to specifically enhance CXCL2 (CXC chemokine ligand 2) expression . This site-specific phosphorylation represents one of the mechanisms through which cells can fine-tune inflammatory responses by regulating a subset of NF-κB target genes.

How should I validate the specificity of a Phospho-RELA (Thr435) antibody for my research?

To validate the specificity of a Phospho-RELA (Thr435) antibody, implement the following methodological approach:

• Phosphatase treatment control: Treat half of your sample with lambda phosphatase before immunoblotting to demonstrate phospho-specificity.

• Blocking peptide competition: Perform parallel Western blots with antibody pre-incubated with phospho-peptide versus non-phospho-peptide .

• Phosphomimetic mutants: Use cells expressing T435D (phosphomimetic) and T435A (phospho-null) RelA mutants as positive and negative controls .

• Stimulus-dependent phosphorylation: Verify increased phospho-signal after TNFα treatment, as this has been shown to induce Thr435 phosphorylation .

• Knockdown/knockout validation: Use RelA-null cells as negative controls to confirm antibody specificity .

Commercial antibodies should be validated with these approaches, as demonstrated in immunohistochemistry and Western blot analyses showing signal elimination when blocked with phospho-peptide .

What applications are most suitable for Phospho-RELA (Thr435) antibodies in research?

Based on validated applications, Phospho-RELA (Thr435) antibodies are most effectively employed in the following research applications:

For ChIP applications, this antibody is particularly valuable for investigating how Thr435 phosphorylation affects RelA binding to specific gene promoters and the subsequent recruitment of transcriptional cofactors. When performing Western blot analysis, include both total RelA and phospho-specific antibodies to normalize phosphorylation levels to total protein expression .

How does Thr435 phosphorylation mechanistically affect RelA transcriptional activity?

The mechanistic impact of Thr435 phosphorylation on RelA transcriptional activity involves complex protein-protein interactions that affect chromatin remodeling:

• HDAC1 Interaction: Mutation studies have demonstrated that Thr435 phosphorylation disrupts RelA interaction with HDAC1 in vitro . This disruption appears to be direct and specific to this phosphorylation site.

• Histone Acetylation: ChIP analysis revealed that the T435D phosphomimetic mutation results in enhanced levels of histone acetylation at NF-κB target gene promoters . This is consistent with decreased HDAC1 recruitment to these regions.

• Transcriptional Outputs: The effects are gene-specific, with the phosphomimetic T435D mutation significantly enhancing Cxcl2 mRNA levels in reconstituted Rela−/− mouse embryonic fibroblasts .

• TAD Activity Modulation: When tested as a Gal4-TAD fusion protein, the phospho-null T435A mutation dramatically increased transcriptional activity, while the phosphomimetic T435D mutation decreased activity . This suggests that in the isolated TAD context, phosphorylation may repress transcriptional potential.

These seemingly contradictory findings between full-length RelA and the isolated TAD underscore the complex, context-dependent nature of this phosphorylation event in regulating transcriptional outcomes.

What experimental conditions optimize detection of TNFα-induced Thr435 phosphorylation?

Optimal detection of TNFα-induced Thr435 phosphorylation requires specific experimental conditions:

• Cell Type Selection: Mouse embryonic fibroblasts (MEFs) show more robust and rapid Thr435 phosphorylation following TNFα stimulation compared to U-2 OS cells, which exhibit weaker induction . Consider using MEFs for initial studies.

• TNFα Concentration and Timing: Based on published protocols, use 10-20 ng/ml TNFα and monitor phosphorylation between 5-60 minutes post-stimulation .

• Phosphatase Inhibition: Include phosphatase inhibitors (e.g., calyculin A) in cell lysis buffers to preserve phosphorylation . The study demonstrates that calyculin A treatment results in increasing levels of phosphorylation at this site.

• Immunoprecipitation Step: Direct Western blots may show background bands that mask the phospho-specific signal. An immunoprecipitation step with anti-RelA antibody followed by immunoblotting with the phospho-specific antibody can significantly enhance detection sensitivity .

• Nuclear Fraction Enrichment: Since active RelA translocates to the nucleus, analyzing nuclear fractions can enhance detection of the phosphorylated form.

• Sample Preparation: For optimal results, process samples rapidly on ice to prevent dephosphorylation by cellular phosphatases.

How can I investigate the relationship between Thr435 phosphorylation and gene-specific transcriptional responses?

To comprehensively investigate how Thr435 phosphorylation affects gene-specific transcriptional responses, implement the following experimental workflow:

• Generate Stable Cell Lines: Establish Rela−/− cells reconstituted with wild-type RelA, T435A (phospho-null), or T435D (phosphomimetic) mutants as performed in the key study .

• Transcriptional Profiling: Perform RNA-seq or targeted gene expression analysis of NF-κB target genes across these cell lines. Key genes to examine include:

  • Cxcl2 (demonstrated to be enhanced by T435D)

  • Cxcl1 (chemokine with similar regulation)

  • Tnfaip3 (A20, important negative feedback regulator)

• ChIP Analysis: Conduct ChIP experiments using the following antibodies:

  • Anti-RelA

  • Anti-phospho-Thr435 RelA

  • Anti-HDAC1

  • Anti-acetyl histone H3 and H4

• Promoter Analysis: Use the ChIP primers described in the study :

  • Cxcl1 (FWD 5′-CTAATCCTTGGGAGTGGAG-3′, REV 5′-CCCTTTTATGCTCGAAAC-3′)

  • Cxcl2 (FWD 5′-CGTGCATAAAAGGAGCTCTC-3′, REV 5′-GTGCCCGAGGAAGCTTGT-3′)

  • Tnfaip3 (FWD 5′-CGCTGAGAGAGAGACAAAC-3′, REV 5′-TGGCCCTGAAGATTAACT-3′)

• Protein-Protein Interaction Studies: Perform co-immunoprecipitation experiments to assess how Thr435 phosphorylation affects RelA interactions with transcriptional cofactors beyond HDAC1.

This integrated approach will provide mechanistic insights into how this specific phosphorylation event contributes to gene-specific transcriptional regulation.

What kinases are responsible for Thr435 phosphorylation, and how can they be experimentally manipulated?

While the specific kinases responsible for Thr435 phosphorylation aren't explicitly identified in the provided search results, a methodological approach to identify and manipulate these kinases would include:

• Kinase Prediction Analysis: Use bioinformatic tools to analyze the amino acid sequence surrounding Thr435 (TQAGEGT*LSEALC) for potential kinase consensus motifs.

• Kinase Inhibitor Screening: Systematically test the effects of specific kinase inhibitors on TNFα-induced Thr435 phosphorylation. Focus on kinases known to be activated in the TNFα signaling pathway, including:

  • IKK complex members

  • MAP kinases (p38, JNK, ERK)

  • PKC family members

  • GSK3β

• Kinase Overexpression/Knockdown: Overexpress constitutively active forms or perform siRNA knockdown of candidate kinases to assess their impact on Thr435 phosphorylation.

• In Vitro Kinase Assays: Perform in vitro kinase assays using purified candidate kinases and RelA peptides containing the Thr435 site.

• Phosphatase Studies: The research indicates that PP4 (protein phosphatase 4)-mediated Thr435 dephosphorylation enhances RelA-mediated activation following cisplatin treatment . This suggests that targeting PP4 could be an alternative approach to modulate Thr435 phosphorylation levels.

This systematic approach would help identify the relevant kinases and provide tools for experimental manipulation of Thr435 phosphorylation in various research contexts.

How does Thr435 phosphorylation compare to other phosphorylation sites on RelA in terms of functional outcomes?

RelA contains multiple phosphorylation sites that collectively create a complex regulatory network. A comparative analysis of Thr435 phosphorylation with other sites reveals:

The research on Thr435 phosphorylation reveals a unique aspect of RelA regulation where phosphorylation at this site appears to have gene-specific effects rather than globally affecting RelA activity . This contrasts with some other sites (particularly Ser536 and Ser276) that tend to have broader effects on RelA transcriptional capacity.

The context-dependent nature of Thr435 phosphorylation is evident in the observation that a T435A mutation dramatically increases transcriptional activity when tested in the isolated TAD, yet the T435D phosphomimetic enhances specific gene expression in the context of full-length RelA . This suggests that Thr435 phosphorylation operates within a complex network of modifications that collectively determine RelA function in a gene-specific manner.

What are common pitfalls when working with Phospho-RELA (Thr435) antibodies and how can they be avoided?

When working with Phospho-RELA (Thr435) antibodies, researchers frequently encounter several challenges. Here are methodological solutions to common pitfalls:

• Background Signal Issues:

  • Problem: Direct Western blots may show background bands that mask the phospho-specific signal .

  • Solution: Implement an immunoprecipitation step with anti-RelA antibody before immunoblotting with the phospho-specific antibody .

• Phosphorylation Loss During Sample Processing:

  • Problem: Rapid dephosphorylation by cellular phosphatases.

  • Solution: Include phosphatase inhibitors (e.g., calyculin A) in all buffers and process samples rapidly on ice .

• Cell Type Variability:

  • Problem: Variable detection of Thr435 phosphorylation across cell types.

  • Solution: The research indicates that MEFs show more robust phosphorylation than U-2 OS cells following TNFα stimulation . Select appropriate cell types for initial studies.

• Antibody Cross-Reactivity:

  • Problem: Non-specific binding to other phosphorylated proteins.

  • Solution: Always validate specificity using blocking peptides and phospho-null mutants (T435A) .

• Fixation-Induced Epitope Masking in IHC:

  • Problem: Formalin fixation can mask phospho-epitopes.

  • Solution: Optimize antigen retrieval methods; validated IHC protocols indicate successful detection in paraffin-embedded tissues .

• Quantification Challenges:

  • Problem: Difficulty normalizing phospho-signal to total RelA.

  • Solution: Always run parallel blots or sequential probing for total RelA and calculate the phospho/total ratio.

What sample preparation methods best preserve Thr435 phosphorylation for different analytical techniques?

Optimal preservation of Thr435 phosphorylation requires technique-specific sample preparation methods:

The research specifically demonstrates that immunoprecipitation with an anti-RelA antibody followed by immunoblotting with the phospho-specific antibody significantly enhances detection sensitivity compared to direct Western blots . This approach should be considered when working with challenging samples or when signal-to-noise ratio is problematic.

How might single-cell analysis techniques advance our understanding of RelA Thr435 phosphorylation dynamics?

Single-cell analysis presents transformative opportunities for studying the heterogeneity and dynamics of RelA Thr435 phosphorylation:

• Single-Cell Phospho-Flow Cytometry:

  • Implementation of phospho-specific antibodies in flow cytometry would allow quantification of phosphorylation levels across thousands of individual cells.

  • This approach could reveal cell-to-cell variability in TNFα responses that may be masked in population-based analyses.

• Single-Cell RNA-Seq Integration:

  • Combining phospho-flow with single-cell RNA-seq would allow correlation between Thr435 phosphorylation states and gene expression profiles at the single-cell level.

  • This could reveal subpopulations of cells with distinct phosphorylation states and corresponding transcriptional outputs.

• Live-Cell Phosphorylation Sensors:

  • Development of FRET-based biosensors incorporating the RelA region around Thr435 would enable real-time visualization of phosphorylation dynamics in living cells.

  • This approach could reveal the temporal dynamics of Thr435 phosphorylation and dephosphorylation following TNFα stimulation with unprecedented resolution.

• Spatial Proteomics:

  • Mass spectrometry imaging or proximity ligation assays could map the subcellular localization of phosphorylated RelA at Thr435.

  • This would provide insights into whether Thr435 phosphorylation occurs predominantly in specific nuclear regions or protein complexes.

These single-cell approaches would address limitations of current bulk analyses and potentially reveal new regulatory mechanisms governing the specificity of NF-κB transcriptional responses.

What therapeutic implications does targeting RelA Thr435 phosphorylation have for inflammatory diseases?

The gene-specific effects of Thr435 phosphorylation suggest potential therapeutic applications:

• Selective Inflammatory Gene Modulation:

  • Since Thr435 phosphorylation specifically enhances expression of certain genes (e.g., Cxcl2) while not globally affecting NF-κB activity, targeting this modification could provide more selective anti-inflammatory effects compared to broad NF-κB inhibitors.

• Identification of Pathway-Specific Inhibitors:

  • Targeting the kinases responsible for Thr435 phosphorylation could provide more selective therapeutic options than targeting upstream NF-κB pathway components.

  • Alternatively, inhibitors that disrupt the structural changes induced by Thr435 phosphorylation could be developed.

• Biomarker Potential:

  • Phospho-Thr435 levels could serve as biomarkers for specific inflammatory states or disease progression.

  • The available antibodies could be adapted for clinical diagnostic applications.

• Combined Epigenetic Therapy Approaches:

  • Since Thr435 phosphorylation affects HDAC1 recruitment and histone acetylation , combination therapies targeting both this phosphorylation site and histone-modifying enzymes might provide synergistic anti-inflammatory effects.

Future research should focus on identifying the specific inflammatory conditions where Thr435 phosphorylation plays a critical role, as this would determine the most promising therapeutic applications.