Phospho-RELA (T435) Antibody

What is the biological significance of RelA Thr435 phosphorylation?

Thr435 phosphorylation represents a key regulatory mechanism that modulates the transcriptional activity of the RelA (p65) subunit of NF-κB. This phosphorylation occurs within the C-terminal transactivation domain (TAD) of RelA and has been demonstrated to have gene-specific effects on transcription . Experimental evidence using mutational analysis reveals that phosphorylation at this site can both enhance and repress transcriptional activity depending on the target gene context. For instance, a T435D phosphomimetic mutant significantly enhances Cxcl2 (CXC chemokine ligand 2) mRNA levels in reconstituted Rela −/− mouse embryonic fibroblasts .

The phosphorylation at Thr435 provides an additional mechanism for modulating the specificity of NF-κB transcriptional activity in cells. Mechanistically, this modification appears to alter the interaction between RelA and transcriptional co-regulators, particularly histone deacetylase 1 (HDAC1), thereby affecting chromatin remodeling at target gene promoters . This phosphorylation event thus represents a sophisticated mechanism for fine-tuning inflammatory and immune responses at the transcriptional level.

How is Phospho-RELA (T435) detected in experimental settings?

Detection of Phospho-RELA (T435) relies primarily on phospho-specific antibodies that have been validated to recognize this specific modification. Several approaches can be employed:

• Western Blotting: Phospho-RELA (T435) can be detected via western blot using specific antibodies. Due to background bands, immunoprecipitation with an anti-RelA antibody followed by western blotting with the phospho-specific antibody often yields clearer results . The observed molecular weight is typically around 58-60 kDa .

• Immunoprecipitation: When background signal is problematic, immunoprecipitation with a general RelA antibody followed by probing with the phospho-specific antibody improves detection .

• Chromatin Immunoprecipitation (ChIP): Phospho-RELA (T435) antibodies can be used in ChIP assays to detect the presence of phosphorylated RelA at specific promoters. Researchers have used this approach with primers for genes like Cxcl1, Cxcl2, and Tnfaip3 .

• Immunohistochemistry: Phospho-RELA (T435) antibodies have been validated for immunohistochemistry on paraffin-embedded tissues, enabling visualization of the phosphorylated protein in tissue sections .

For optimal results, stimulation with TNFα or phosphatase inhibitors like calyculin A enhances phosphorylation levels and improves detection .

What are the practical considerations for antibody selection and validation?

When selecting and validating Phospho-RELA (T435) antibodies, researchers should consider:

• Antibody Specificity: Ensure the antibody specifically recognizes the phosphorylated form of Thr435. ELISA analysis with phospho- and non-phospho-peptides can confirm this specificity . Commercially available antibodies are typically raised against synthetic phosphopeptides with sequences surrounding Thr435 (e.g., E-G-T(p)-L-S) .

• Host Species and Reactivity: Available antibodies are predominantly rabbit polyclonal with reactivity to human, mouse, and rat RELA . This cross-reactivity enables comparative studies across species.

• Validation Methods: Look for antibodies validated through multiple applications (Western blot, ELISA, IHC-P) and with positive controls such as TNFα-treated cells or phosphatase inhibitor-treated samples .

• Working Dilutions: Typical recommended dilutions for Western blotting range from 1:500 to 1:2000 .

• Positive Controls: NIH/3T3 cells or HeLa cells treated with TNFα are commonly used as positive controls for antibody validation .

The inclusion of appropriate controls is critical - comparisons between untreated and TNFα+CA (calyculin A) treated samples can verify the specificity and functionality of the antibody .

How does Thr435 phosphorylation affect RelA interactions with transcriptional machinery?

Thr435 phosphorylation significantly alters RelA's protein-protein interactions with transcriptional regulators, particularly those involved in chromatin remodeling. Research indicates that:

• HDAC1 Interaction: Mutation of Thr435 disrupts RelA interaction with histone deacetylase 1 (HDAC1) in vitro . This suggests that phosphorylation at this site modulates HDAC1 recruitment to NF-κB target gene promoters.

• Histone Acetylation Levels: Chromatin immunoprecipitation analysis reveals that the T435D phosphomimetic mutation results in enhanced levels of histone acetylation, consistent with decreased recruitment of HDAC1 . This mechanistically explains how this phosphorylation can affect chromatin accessibility and gene expression.

• Transcriptional Consequences: The effects on transcription are gene-specific. For instance, a T435A phospho-null mutation dramatically increases transcriptional activity in reporter assays, while the T435D phosphomimetic mutation decreases activity . This suggests that phosphorylation at this site may generally repress the transcriptional potential of the RelA TAD, but with context-dependent outcomes.

• Domain-Specific Effects: When investigating exclusively TAD-dependent effects using Gal4-DBD fusions with the RelA TAD, the T435A mutation significantly enhances transcriptional activity while T435D decreases it . This confirms that the modifications directly affect TAD functionality.

These findings collectively demonstrate that Thr435 phosphorylation serves as a molecular switch that modulates RelA's ability to interact with transcriptional co-regulators, particularly those involved in histone modifications.

What experimental approaches can resolve temporal dynamics of Thr435 phosphorylation?

The temporal dynamics of Thr435 phosphorylation present specific experimental challenges that require sophisticated approaches:

• Time-Course Stimulation: Studies show different temporal patterns depending on cell type. In U-2 OS cells, TNFα induces weak phosphorylation, while in MEF cells, rapid Thr435 phosphorylation is detected following TNFα stimulation . Experiments should include multiple time points (e.g., 5, 10, and 20 minutes post-stimulation) to capture the transient nature of this modification .

• Phosphatase Inhibition: Treatment with serine/threonine phosphatase inhibitors like calyculin A results in increasing levels of phosphorylation at Thr435 . This approach can help stabilize the phosphorylation for detection purposes.

• Combined Immunoprecipitation and Western Blotting: Due to background bands in direct Western blots, immunoprecipitation with an anti-RelA antibody followed by probing with the phospho-specific antibody provides clearer visualization of temporal dynamics .

• Promoter-Specific ChIP Analysis: Thr435 phosphorylation of promoter-bound RelA can be detected at specific NF-κB target genes following TNFα treatment. Using primers for genes like Cxcl1, Cxcl2, and Tnfaip3 allows for monitoring of gene-specific temporal patterns .

• Quantitative Phosphoproteomics: Mass spectrometry-based approaches can provide unbiased assessment of phosphorylation dynamics, although this requires appropriate enrichment strategies for phosphopeptides.

When designing temporal studies, it's important to consider that different cell types may exhibit distinct phosphorylation kinetics in response to the same stimulus .

How does Thr435 phosphorylation integrate with other post-translational modifications of RelA?

RelA undergoes numerous post-translational modifications that collectively form a complex regulatory code. Understanding how Thr435 phosphorylation integrates with other modifications requires consideration of:

• Modification Crosstalk: Phosphorylation of sites within the TAD of RelA leads to both increased and decreased levels of transcriptional activity, with the precise effect dependent on context and gene target . The interplay between Thr435 phosphorylation and other modifications (phosphorylation, acetylation, methylation, ubiquitination) remains an area requiring further investigation.

• Phosphorylation-Dephosphorylation Cycles: PP4 (protein phosphatase 4)-mediated Thr435 dephosphorylation was previously proposed to enhance RelA-mediated activation following cisplatin treatment, suggesting that phosphorylation at this site can negatively affect RelA activity in specific contexts . This highlights the importance of considering both kinases and phosphatases in the regulatory cycle.

• Functional Consequences: While some modifications enhance transcriptional activity, others may repress it or affect protein stability and degradation . Creating a comprehensive map of how these modifications interact requires systematic mutation studies and mass spectrometry analysis.

• Spatial Considerations: RelA localizes to both cytoplasm and nucleus , and different modifications may occur preferentially in different cellular compartments. Fractionation studies combined with phospho-specific detection can help resolve these spatial dynamics.

• Stimulus-Specific Responses: Different stimuli (TNFα, cisplatin, etc.) may induce distinct patterns of post-translational modifications . Comparative studies across multiple stimuli can help elucidate these patterns.

Understanding this modification "code" remains challenging but is essential for developing targeted interventions in NF-κB-related diseases.

What are the technical considerations for using Phospho-RELA (T435) antibodies in chromatin immunoprecipitation?

Chromatin immunoprecipitation (ChIP) with Phospho-RELA (T435) antibodies requires specific technical considerations:

• Primer Design: For successful ChIP experiments, researchers have used specific primers for NF-κB target genes:

  • 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′

• Antibody Specificity and Validation: Due to the transient nature of this phosphorylation, antibody specificity is critical. Control experiments should include IgG controls, input controls, and ideally, comparisons with ChIP using general RelA antibodies .

• Stimulus Conditions: TNFα treatment induces Thr435 phosphorylation of promoter-bound RelA at NF-κB target genes . Optimizing stimulus conditions (concentration, timing) is essential for successful ChIP experiments.

• Sequential ChIP: To determine if RelA phosphorylated at Thr435 is associated with specific co-factors or histone modifications, sequential ChIP (re-ChIP) approaches may be employed, immunoprecipitating first with anti-RelA and then with anti-phospho-Thr435 antibodies.

• Quantification Methods: qPCR is typically used to quantify ChIP results, requiring careful normalization against input DNA and selection of appropriate reference genes or regions .

These technical considerations are essential for generating reliable and reproducible data when investigating the genomic binding patterns of Phospho-RELA (T435).

How can Phospho-RELA (T435) antibodies be utilized in studying disease mechanisms?

Phospho-RELA (T435) antibodies offer valuable tools for investigating disease mechanisms, particularly in conditions involving dysregulated inflammation and immune responses:

• Cancer Research: These antibodies have been successfully used in cancer studies, including breast carcinoma tissue analysis via immunohistochemistry . The NF-κB pathway plays crucial roles in cancer-related inflammation and cell survival, making this phosphorylation potentially relevant to cancer progression.

• Inflammatory Diseases: Given the role of NF-κB in inflammatory processes, studying Thr435 phosphorylation may provide insights into conditions like rheumatoid arthritis, inflammatory bowel disease, and asthma. The gene-specific effects of this modification on inflammatory mediators like Cxcl2 are particularly relevant .

• Mechanistic Studies: In disease models, these antibodies can help elucidate how signaling pathways converge on NF-κB regulation. For example, researchers have used these antibodies in studies of oxidative stress, as demonstrated in work by Tian Li et al. using MLE-12 cells .

• Therapeutic Response Monitoring: Since NF-κB is a target for various therapeutic interventions, monitoring Thr435 phosphorylation could potentially serve as a biomarker for treatment efficacy.

• Tissue-Specific Analyses: The antibodies' validated use in immunohistochemistry enables analysis of tissue-specific patterns of RelA phosphorylation in disease states .

When designing disease-focused studies, researchers should consider the cell type-specific and stimulus-specific nature of Thr435 phosphorylation, as these patterns may vary considerably across different pathological contexts .

What are the optimal conditions for inducing and detecting Thr435 phosphorylation?

Optimizing experimental conditions for reliable detection of Thr435 phosphorylation requires attention to several factors:

• Stimulation Protocols:

  • TNFα treatment: Effective concentrations range from 5 ng/ml to 20 ng/ml, with phosphorylation detectable as early as 5 minutes post-stimulation in some cell types .

  • Phosphatase inhibition: Treatment with calyculin A (a serine/threonine phosphatase inhibitor) results in increasing levels of phosphorylation at Thr435 and can be used to enhance detection .

• Cell Type Considerations:

  • MEF cells show rapid Thr435 phosphorylation following TNFα stimulation .

  • U-2 OS cells show weak induction of phosphorylation at this site following TNFα stimulation .

  • HeLa cells treated with TNFα show detectable phosphorylation and serve as good positive controls .

  • NIH/3T3 cells are also used as positive controls for antibody validation .

• Detection Methods:

  • Direct Western blotting may show background bands that can mask the specific signal .

  • Immunoprecipitation with anti-RelA antibody followed by Western blotting with the phospho-specific antibody yields clearer results .

  • For Western blotting, recommended antibody dilutions range from 1:500 to 1:2000 .

• Sample Preparation:

  • Protein loading amount of approximately 30 μg is recommended for Western blot detection .

  • Primary antibody incubation conditions: overnight at 4°C for optimal results .

• Controls:

  • Untreated versus TNFα+CA (calyculin A) treated samples provide important controls .

  • Time-course experiments (5, 10, 20 minutes post-stimulation) help capture the transient nature of the phosphorylation .

These optimized conditions ensure reliable detection of Thr435 phosphorylation across different experimental settings.

How can mutational analysis be used to study the functional significance of Thr435 phosphorylation?

Mutational analysis provides critical insights into the functional significance of Thr435 phosphorylation:

• Phospho-Null and Phospho-Mimetic Mutations:

  • T435A (phospho-null) mutation: Replacing threonine with alanine prevents phosphorylation at this site.

  • T435D (phospho-mimetic) mutation: Replacing threonine with aspartic acid mimics constitutive phosphorylation .

• Expression Systems:

  • Full-length RelA mutants: Allow investigation of effects in the context of the complete protein.

  • TAD-fusion proteins with the DNA-binding domain (DBD) of Gal4: Enable isolation of TAD-dependent effects .

  • Reconstitution in Rela −/− mouse embryonic fibroblasts: Provides a clean genetic background for functional studies .

• Functional Readouts:

  • Reporter assays: T435A mutation dramatically increases transcriptional activity, while T435D mutation decreases activity in U-2 OS cells .

  • Gene expression analysis: T435D phosphomimetic mutant significantly enhances Cxcl2 mRNA levels in reconstituted Rela −/− MEFs .

  • Chromatin immunoprecipitation: T435D mutation results in enhanced levels of histone acetylation associated with decreased recruitment of HDAC1 .

• Protein Interaction Studies:

  • In vitro binding assays: Mutation of Thr435 disrupts RelA interaction with HDAC1 .

  • Co-immunoprecipitation: Can be used to validate altered interactions in cellular contexts.

• Experimental Design Considerations:

  • Gene-specific effects: Mutations may have different effects depending on the target gene context .

  • Cell type-specific effects: Responses may vary across different cell types .

This systematic mutational approach has revealed that Thr435 phosphorylation modulates RelA function in a context-dependent manner, affecting both its transcriptional activity and protein interactions.

What are the emerging areas of research involving Phospho-RELA (T435)?

Several promising research directions are emerging in the study of Phospho-RELA (T435):

• Integration with Single-Cell Technologies: Applying single-cell approaches to study cell-to-cell variation in Thr435 phosphorylation patterns could reveal heterogeneity in NF-κB signaling responses that bulk analyses miss.

• Structural Biology Insights: Resolving how Thr435 phosphorylation alters the three-dimensional structure of RelA could provide mechanistic insights into its effects on protein-protein interactions and DNA binding.

• Kinase and Phosphatase Networks: While PP4 has been implicated in Thr435 dephosphorylation , the specific kinases responsible for this phosphorylation remain to be fully characterized. Mapping these regulatory networks would provide new intervention points.

• Therapeutic Targeting: Given the gene-specific effects of Thr435 phosphorylation, developing strategies to selectively modulate this modification could offer more precise approaches to targeting NF-κB in disease.

• Cross-Talk with Non-Canonical NF-κB Pathways: Investigating how Thr435 phosphorylation interfaces with non-canonical NF-κB signaling could reveal additional regulatory mechanisms.

• Tissue-Specific Regulation: Since differential phosphorylation patterns have been observed across cell types , exploring tissue-specific regulation of Thr435 phosphorylation could help explain context-dependent NF-κB functions.

• Roles in Chronic Inflammation: Given the importance of NF-κB in inflammatory processes, understanding how sustained alterations in Thr435 phosphorylation contribute to chronic inflammatory conditions represents an important research direction.

These emerging areas highlight the continuing importance of Phospho-RELA (T435) antibodies as tools for advancing our understanding of NF-κB signaling complexity.

What are the limitations of current Phospho-RELA (T435) antibodies and potential solutions?

Current Phospho-RELA (T435) antibodies face several limitations that researchers should consider:

• Background Signal: Direct Western blotting often shows background bands that can mask the specific signal .

  • Solution: Immunoprecipitation with anti-RelA antibody before Western blotting with phospho-specific antibody improves signal clarity .

• Transient Nature of Phosphorylation: The transient nature of Thr435 phosphorylation can make detection challenging.

  • Solution: Use of phosphatase inhibitors like calyculin A can stabilize the phosphorylated state .

• Cell Type Variability: Different cell types show variable patterns of Thr435 phosphorylation in response to the same stimulus .

  • Solution: Thorough validation in each cell type of interest and optimization of stimulation protocols.

• Specificity Concerns: Ensuring absolute specificity for the phosphorylated form remains challenging.

  • Solution: Two-step peptide affinity chromatography with phospho- and non-phospho-peptides during antibody purification improves specificity .

• Limited Applications: Not all commercially available antibodies are validated for all potential applications.

  • Solution: Comprehensive validation across multiple applications (WB, ELISA, IHC-P, ChIP) would expand utility .

• Reproducibility Issues: Batch-to-batch variation in polyclonal antibodies can affect reproducibility.

  • Solution: Development of monoclonal antibodies or recombinant antibodies could improve consistency.

• Cross-Reactivity: Some antibodies show cross-reactivity with related phosphorylation sites.

  • Solution: Careful validation using phospho-null mutants (T435A) as negative controls.