Phospho-RELA (Thr505) Antibody

What is the biological significance of RelA(p65) Thr505 phosphorylation?

RelA(p65) Thr505 phosphorylation serves as a critical negative regulator of NF-κB function, particularly in response to DNA damage and replication stress. Unlike complete RelA knockout, which is embryonically lethal due to TNF-induced liver apoptosis, the selective mutation of this phosphorylation site (T505A) produces viable mice with specific phenotypes. These include:

• Aberrant hepatocyte proliferation following liver injury or partial hepatectomy

• Earlier onset of hepatocellular carcinoma in chemical induction models

• Enhanced tumor-promoting activities of RelA

This phosphorylation event provides a mechanism to suppress the oncogenic potential of NF-κB signaling, acting as a molecular switch that converts RelA from a pro-survival factor to one that can promote apoptosis under specific conditions .

Which kinases are responsible for RelA Thr505 phosphorylation?

The checkpoint kinase Chk1 has been identified as the primary kinase responsible for phosphorylating RelA at Thr505. This phosphorylation occurs as part of a signaling cascade involving:

• ATR (Ataxia Telangiectasia and Rad3-related protein) activation, which phosphorylates and activates Chk1

• Chk1-mediated phosphorylation of RelA at Thr505

• Subsequent modulation of RelA transcriptional activity

This pathway is specifically activated during S-phase of the cell cycle and in response to DNA replication stress. Importantly, Chk1 exhibits specificity for the Thr505 site, which conforms to the consensus Chk1 phosphorylation sequence .

How is RelA Thr505 phosphorylation regulated during the cell cycle?

RelA Thr505 phosphorylation displays distinct cell cycle-dependent regulation:

• Peaks during S phase when the DNA replication checkpoint is active

• Continues at lower levels in G2 phase

• Shows minimal phosphorylation in G1 phase

This pattern contrasts with other RelA phosphorylation sites; for example, S468 phosphorylation predominates in G1 phase while S536 phosphorylation peaks in G2 phase. This differential phosphorylation creates a temporal code that modulates RelA activity throughout the cell cycle .

An antagonistic relationship exists between Akt and Chk1 signaling pathways in regulating NF-κB activity during the cell cycle:

• Inhibition of Akt1 increases RelA T505 phosphorylation

• Inhibition of Chk1 or ATR decreases RelA T505 phosphorylation

• Aphidicolin (causing S-phase arrest and Chk1 activation) increases T505 phosphorylation

What are the optimal applications for Phospho-RELA (Thr505) antibodies?

Based on commercial antibody specifications and published literature, Phospho-RELA (Thr505) antibodies have been successfully used in multiple applications:

When selecting the appropriate application, consider:

• Western blotting provides information about protein size and relative abundance

• IHC reveals spatial distribution within tissues (e.g., tumor vs. adjacent tissue)

• Immunofluorescence allows co-localization studies with other proteins or cellular structures

How can I validate the specificity of a Phospho-RELA (Thr505) antibody?

Proper validation of phospho-specific antibodies is crucial for reliable results. Recommended validation strategies include:

• Phosphatase treatment control: Treat half of your sample with lambda phosphatase before immunoblotting; the signal should disappear in the treated sample

• Phospho-mimetic and phospho-deficient mutants: Compare antibody reactivity against:

  • Wild-type RelA

  • T505A mutant (phospho-deficient)

  • T505D mutant (phospho-mimetic)

  Published data shows reduced antibody signal with both mutants compared to phosphorylated wild-type protein

• Stimulus-dependent phosphorylation: Test antibody reactivity following treatments known to induce or inhibit T505 phosphorylation:

  • Cisplatin treatment (increases phosphorylation)

  • Chk1 inhibitors like Gö6976 (decreases phosphorylation)

  • ATR inhibitors like caffeine (decreases phosphorylation)

• siRNA knockdown: Reduce RelA expression using siRNA; the phospho-specific signal should decrease proportionally

What experimental models are most suitable for studying RelA Thr505 phosphorylation?

Several experimental models have been successfully employed to study RelA Thr505 phosphorylation:

Cell Line Models:

• U2OS osteosarcoma cells (show inducible T505 phosphorylation after cisplatin)

• RelA-reconstituted rela−/− mouse embryonic fibroblasts (MEFs)

• RelA T505A MEFs derived from knockin mice

Animal Models:

• RelA T505A knockin mice (viable, unlike RelA knockout mice)

• Liver regeneration models (partial hepatectomy)

• Chemical injury models (CCl₄ treatment)

• Hepatocellular carcinoma model (N-nitrosodiethylamine treatment)

• Eμ-Myc lymphoma model crossed with RelA T505A mice

The choice of model depends on your specific research question:

• For biochemical studies of phosphorylation mechanisms: cell lines

• For physiological and pathological relevance: animal models

• For cancer studies: both approaches are complementary

How can I investigate the downstream effects of RelA Thr505 phosphorylation?

To elucidate downstream effects of RelA Thr505 phosphorylation, consider multi-layered approaches:

• Transcriptomic analysis:

  • Compare gene expression profiles between wild-type RelA and T505A mutant cells

  • Focus on known NF-κB target genes and stress-response genes

  • Analyze both upregulated and downregulated genes (T505 phosphorylation has been shown to repress antiapoptotic genes like Bcl-xL)

• Chromatin immunoprecipitation (ChIP):

  • Use phospho-T505 specific antibodies for ChIP-seq to identify genomic binding sites

  • Compare RelA binding profiles between phosphorylated and non-phosphorylated states

  • Examine recruitment of corepressors to RelA target genes (phospho-T505 facilitates corepressor docking)

• Protein interaction studies:

  • Immunoprecipitation with phospho-T505 antibodies to identify interacting partners

  • Compare protein complexes formed by wild-type vs. T505A RelA

  • Focus on interactions with chromatin modifiers and transcriptional regulators

• Cellular phenotype analysis:

  • Assess effects on cell proliferation (T505A increases proliferation)

  • Measure apoptotic responses to DNA-damaging agents

  • Evaluate cellular migration and cytoskeletal changes

  • Quantify autophagy markers (T505 phosphorylation regulates autophagy)

How can I resolve contradictory data regarding RelA Thr505 phosphorylation across different experimental systems?

Discrepancies in RelA Thr505 phosphorylation studies might stem from several factors:

• Cell type-specific effects:

  • Compare primary cells vs. immortalized/cancer cell lines

  • Assess baseline NF-κB activity (constitutively active in some cancer cells)

  • Examine p53 status (influences the apoptotic response to T505 phosphorylation)

  • Consider tissue-specific cofactors that modify RelA function

• Experimental conditions:

  • Standardize inducing stimuli (concentration, duration, treatment protocol)

  • Control cell cycle phase (synchronize cells when possible)

  • Account for crosstalk with other signaling pathways (especially Akt)

• Technical considerations:

  • Use multiple antibodies targeting the same phosphorylation site

  • Include appropriate controls (phosphatase treatment, blocking peptides)

  • Confirm findings using complementary techniques (mass spectrometry)

• Genetic background:

  • When using knockout or knockin models, consider compensatory mechanisms

  • Back-cross to establish consistent genetic backgrounds

  • Use littermate controls to minimize variation

What are the technical challenges in studying temporal dynamics of RelA Thr505 phosphorylation?

Investigating the temporal dynamics of RelA Thr505 phosphorylation presents several challenges:

• Rapid kinetics and transient nature:

  • Phosphorylation events can occur within minutes of stimulus

  • Use fine-grained time course experiments with multiple early timepoints

  • Consider pulse-chase approaches with synchronized cells

• Heterogeneity in cell populations:

  • Single-cell analysis techniques may reveal subpopulations with different phosphorylation patterns

  • Flow cytometry with phospho-specific antibodies can quantify cellular heterogeneity

  • Cell synchronization helps reduce variability but may introduce artifacts

• Signal amplification issues:

  • Low abundance of phosphorylated protein may require signal enhancement

  • Phospho-enrichment methods (phosphopeptide enrichment columns, IMAC)

  • Proximity ligation assays for detecting low-abundance modifications in situ

• Spatial dynamics:

  • RelA shuttles between cytoplasm and nucleus

  • Phospho-T505 RelA predominantly localizes to the nucleus

  • Use subcellular fractionation combined with western blotting

  • High-resolution imaging with phospho-specific antibodies

How does RelA Thr505 phosphorylation interact with other post-translational modifications?

RelA function is regulated by multiple post-translational modifications (PTMs), creating a complex regulatory code:

• Interplay with other phosphorylation sites:

  • RelA contains numerous phosphorylation sites including S468 and S536

  • T505 phosphorylation shows distinct cell cycle regulation compared to S468 and S536

  • Different phosphorylation patterns likely determine transcriptional outcomes

• Crosstalk with other PTM types:

  • Consider acetylation, methylation, ubiquitination, and SUMOylation

  • These modifications may enhance or antagonize T505 phosphorylation effects

  • Multiple modifications may occur simultaneously or sequentially

• Experimental approach:

  • Generate combination mutants (e.g., T505A/S536A double mutant)

  • Mass spectrometry-based analysis of the complete modification landscape

  • Sequential immunoprecipitation with antibodies against different modifications

  • Computational modeling of PTM combinations and their functional outcomes

What is the potential role of RelA Thr505 phosphorylation in cancer therapeutics?

The tumor-suppressive function of RelA T505 phosphorylation suggests several therapeutic applications:

• Sensitization to chemotherapy:

  • T505 phosphorylation enhances cisplatin-induced apoptosis

  • Targeting pathways that promote T505 phosphorylation could sensitize resistant tumors

  • Combination strategies with Chk1 activators and chemotherapeutic agents

• Biomarker potential:

  • T505 phosphorylation status might predict treatment response

  • Lower T505 phosphorylation correlates with more aggressive tumors in mouse models

  • Immunohistochemical analysis using phospho-T505 antibodies could stratify patients

• Targeted therapy approaches:

  • Modulating the balance between pro-survival and pro-apoptotic NF-κB functions

  • Small molecules that mimic T505 phosphorylation effects

  • PROTAC approaches targeting non-phosphorylated RelA for degradation

How can single-cell analysis enhance our understanding of RelA Thr505 phosphorylation dynamics?

Single-cell technologies offer new insights into the heterogeneity of RelA T505 phosphorylation:

• Single-cell phosphoproteomics:

  • Reveals cell-to-cell variability in phosphorylation levels

  • Can identify rare subpopulations with distinct phosphorylation patterns

  • Correlates phosphorylation with cellular phenotypes

• Live-cell imaging approaches:

  • Phospho-specific fluorescent biosensors

  • FRET-based reporters for real-time monitoring

  • Correlative light and electron microscopy for ultrastructural localization

• Single-cell transcriptomics:

  • Links phosphorylation states to transcriptional outputs

  • Identifies gene expression signatures associated with T505 phosphorylation

  • Helps define the cellular consequences of phosphorylation heterogeneity

Current evidence suggests significant heterogeneity in NF-κB responses even within genetically identical populations, which may have important implications for understanding differential responses to therapies targeting this pathway .