Acetyl-RELA (K314/K315) Antibody

What is RELA/p65 and why is its acetylation important in research?

RELA (also known as p65) is a subunit of the nuclear factor kappaB (NF-κB) transcription factor that plays a crucial role in regulating genes involved in immunity, cell survival, proliferation, and differentiation. The acetylation of RELA represents an important post-translational modification that regulates NF-κB activity in the nucleus, determining both the duration and strength of NF-κB nuclear activity as well as its transcriptional output . This makes studying RELA acetylation essential for understanding the fine-tuning of inflammatory and immune responses at the molecular level. Researchers investigating cellular signaling pathways, inflammation, or immune regulation would find RELA acetylation a critical research target.

What specific lysine residues on RELA can be acetylated?

Seven acetylated lysines have been identified within RelA/p65, including lysines 122, 123, 218, 221, 310, 314, and 315 . The majority of these lysines are acetylated by p300/CBP, although some, such as K122 and K123, can also be acetylated by PCAF . Each acetylation site has distinct functional consequences on NF-κB activity, making the study of site-specific acetylation particularly important in understanding the nuanced regulation of NF-κB-dependent gene expression.

What is the specific functional relevance of K314/K315 acetylation?

Unlike acetylation at other lysine residues of RELA/p65, acetylation at K314 and K315 by p300 does not affect NF-κB shuttling, DNA binding capabilities, or the induction of anti-apoptotic genes . Instead, K314/K315 acetylation differentially regulates the expression of specific sets of NF-κB target genes in response to TNF-α stimulation . This site-specific acetylation represents a unique regulatory mechanism that allows for selective gene expression control, making it a fascinating target for researchers studying the specificity of NF-κB-dependent transcriptional programs.

What methods can be used to detect RELA K314/K315 acetylation?

Several methods can be employed to detect RELA K314/K315 acetylation:

• Immunoblotting with site-specific antibodies: Using monoclonal antibodies specifically developed against acetylated K314/K315 sites (such as those described in search results #4 and #5) .

• In vitro acetylation assays: These can be performed using recombinant RELA protein, p300 as the acetylating enzyme, and either radioactive [14C]-acetyl-CoA or non-radioactive acetyl-CoA as donors .

• Mass spectrometry: MS/MS analysis following tryptic digestion of acetylated RELA can precisely identify acetylation sites .

• Immunoprecipitation followed by western blotting: This approach allows for detection of acetylated RELA from cell extracts, using pan acetyl-lysine antibodies or site-specific antibodies .

The choice of method depends on the specific research question, available resources, and the needed level of sensitivity and specificity.

How can I optimize in vitro acetylation assays for RELA K314/K315?

For optimal in vitro acetylation of RELA K314/K315, consider the following methodological approach:

• Source of HAT enzyme: Commercial GST-p300 HAT domain fusion proteins may not efficiently acetylate recombinant RELA in vitro. Instead, use p300 immunoprecipitated from transfected HEK293T cells as the HAT enzyme source .

• Reaction conditions:

  • Use 1 μg of recombinant RELA protein

  • Add immunoprecipitated p300

  • Include 2 μl of [14C]-acetyl-CoA or acetyl-CoA

  • Prepare in HAT assay buffer

  • Incubate at 30°C for 1 hour

• Detection methods:

  • For radioactive assays: Run SDS-PAGE, fix the gel, enhance with fluorographic reagent, dry, and expose to X-ray film at -70°C to -80°C

  • For non-radioactive assays: Transfer to nitrocellulose membrane and detect with anti-acetylated lysine antibody

This approach maximizes the chances of detecting specific acetylation at K314/K315 residues.

How should I detect acetylation of RELA in cultured cells?

To detect acetylation of RELA in cultured cells, the following protocol is recommended:

• Transfection: Co-express tagged RELA (e.g., T7-tagged or Myc-tagged) with p300 in HEK293T cells .

• Treatment options: For enhanced acetylation detection, treat cells with:

  • HDAC inhibitors (e.g., 2 μM TSA, 5 mM NAM) to prevent deacetylation

  • TNFα (30 ng/ml) to stimulate the NF-κB pathway

  • Combination of both for optimal results

• Extract preparation: Prepare whole cell extracts using appropriate buffers containing protease inhibitors and HDAC inhibitors .

• Immunoprecipitation: Use antibodies against the tag (e.g., anti-myc) to immunoprecipitate RELA .

• Detection: Analyze immunocomplexes by western blotting using anti-acetylated lysine antibodies, followed by reprobing with anti-tag antibodies to confirm equal loading .

This method allows for specific detection of acetylated RELA in a cellular context where the protein undergoes physiological regulation.

How can I distinguish between the functional effects of K314 versus K315 acetylation?

Distinguishing between the individual contributions of K314 and K315 acetylation requires careful experimental design:

• Site-directed mutagenesis: Generate specific mutants including:

  • K314R (maintains K315 integrity)

  • K315R (maintains K314 integrity)

  • K314/315R double mutant (negative control)

  • Wild-type RELA (positive control)

• Genetic complementation: Introduce these constructs into RELA/p65-deficient cells to avoid interference from endogenous RELA .

• Functional analysis: Compare the effects of each mutant on:

  • Gene expression profiles using microarray analysis

  • Specific target gene activation using reporter assays

  • Protein-protein interactions through co-immunoprecipitation studies

In research by Buerki et al., genetic complementation with specific K314R and K315R mutants revealed that acetylation at these sites regulates distinct gene sets differently from wild-type RELA, with some genes being stimulated and others repressed by the acetylation-deficient mutants .

What is the interplay between acetylation at K314/K315 and other post-translational modifications?

The interplay between K314/K315 acetylation and other modifications represents a complex regulatory network:

• Acetylation-methylation crosstalk: Acetylation of K310 can prevent methylation at K314/K315, which is known to negatively regulate NF-κB function by inducing RELA degradation .

• Modification patterns: Consider analyzing how different stimuli might induce specific patterns of multiple modifications rather than focusing on single sites.

• Sequential analysis: To study interplay between modifications, researchers should:

  • Use antibodies specific for each modification

  • Employ mass spectrometry to identify all modifications simultaneously

  • Conduct time-course experiments to determine the sequence of modification events

  • Utilize specific inhibitors for each type of modifying enzyme to establish dependency relationships

This comprehensive approach helps elucidate how various post-translational modifications work together to fine-tune RELA/p65 activity.

How can I identify the specific gene targets regulated by K314/K315 acetylation?

To identify gene targets specifically regulated by K314/K315 acetylation, implement the following methodological approach:

• Genetic complementation system: Use RELA/p65-deficient cells reconstituted with either wild-type RELA or acetylation-deficient mutants (K314R, K315R, or K314/315R) .

• Genome-wide expression analysis: Perform microarray analysis after TNFα treatment to identify differentially regulated genes .

• Validation strategies:

  • Confirm microarray results using qRT-PCR for selected targets

  • Perform ChIP assays to determine RELA binding to promoters of candidate genes

  • Use reporter gene assays to verify transcriptional regulation

• Bioinformatic analysis: Group regulated genes into functional categories and analyze promoter sequences for common regulatory elements.

Previous research has demonstrated that acetylation-deficient mutants of K314/K315 can either stimulate or repress specific genes compared to wild-type RELA, indicating that these acetylation sites contribute to the specificity of NF-κB-dependent gene expression .

What are the critical validation steps for Acetyl-RELA(K314/K315) antibodies?

When using Acetyl-RELA(K314/K315) antibodies, thorough validation is essential:

• Specificity testing:

  • Compare antibody reactivity between wild-type and K314/315R mutant RELA

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

  • Confirm signal reduction after treatment with deacetylases

• Sample controls:

  • Positive control: RELA co-expressed with p300 plus HDAC inhibitors

  • Negative control: RELA expressed alone without p300

  • Mutant control: K314/315R RELA mutant co-expressed with p300

• Cross-reactivity assessment:

  • Test against other acetylated lysines on RELA (K218, K221, K310)

  • Evaluate reactivity across species if working with non-human models

These validation steps ensure that experimental results accurately reflect K314/K315 acetylation status.

What are the optimal applications for Acetyl-RELA(K314/K315) antibodies?

Based on the available information, Acetyl-RELA(K314/K315) antibodies are suitable for several research applications:

• Immunohistochemistry (IHC): Both available antibodies are validated for IHC applications, with recommended dilutions of 1:100-200 .

• ELISA: Antibodies are suitable for enzyme-linked immunosorbent assays .

• Western blotting: Though not explicitly mentioned in the search results, the antibodies likely work for western blot applications given their specificity for acetylated residues.

• Species reactivity: The antibodies show cross-reactivity with human, mouse, and rat samples, making them versatile for comparative studies across these species .

When designing experiments, consider that these antibodies are typically provided in PBS with 0.02% sodium azide and 50% glycerol (pH 7.4) and should be stored at -20°C, avoiding repeated freeze-thaw cycles .

What are common challenges when detecting RELA K314/K315 acetylation?

Researchers may encounter several challenges when working with Acetyl-RELA(K314/K315):

• Low signal strength: Acetylation is often a transient modification affecting only a fraction of the total RELA pool. To address this:

  • Use HDAC inhibitors (2 μM TSA, 5 mM NAM) during cell treatment

  • Co-express p300 to enhance acetylation levels

  • Enrich for nuclear fractions where active RELA is located

• Background issues: Non-specific binding can complicate interpretation. Solutions include:

  • Optimizing blocking conditions with BSA or non-fat milk

  • Including appropriate negative controls (K314/315R mutants)

  • Using more stringent washing conditions

• Temporal dynamics: Acetylation may occur in a narrow time window. Consider:

  • Performing detailed time-course experiments after stimulation

  • Using pulse-chase approaches to track acetylation/deacetylation kinetics

• Protein abundance: Low expression of RELA can limit detection. Consider:

  • Concentrating samples through immunoprecipitation before analysis

  • Using more sensitive detection methods like chemiluminescence

Addressing these challenges systematically will improve detection of K314/K315 acetylation.

How do cell type and stimulation conditions affect RELA K314/K315 acetylation?

RELA K314/K315 acetylation can vary significantly based on cell type and stimulation conditions:

• Cell type considerations:

  • Different cell types may express varying levels of acetyltransferases (p300/CBP) and deacetylases

  • Basal acetylation levels may differ between immune cells, epithelial cells, and other cell types

  • The regulatory machinery controlling acetylation may have tissue-specific components

• Stimulation parameters:

  • TNFα has been shown to induce RELA acetylation and is commonly used at 30 ng/ml

  • Duration of stimulation affects acetylation patterns (typically 30-45 minutes for initial studies)

  • Combined treatment with HDAC inhibitors enhances detection of acetylation

• Experimental recommendations:

  • Optimize stimulation conditions for each cell type

  • Include time-course experiments to identify peak acetylation

  • Consider using multiple stimuli (e.g., TNFα, IL-1β, LPS) for comprehensive analysis

Understanding these variables is crucial for experimental design and interpretation of results across different cellular systems.