Phospho-HDAC3 (Ser424) Antibody

What is HDAC3 and what role does phosphorylation at Ser424 play in its function?

HDAC3 (Histone Deacetylase 3) is a nuclear and cytoplasmic protein that deacetylates both histone substrates (H2A, H3, H4) and non-histone substrates including RelA, SRY, p53, MEF2, PCAF, and p300/CBP. HDAC3 plays a crucial role in transcriptional repression and epigenetic regulation through its deacetylase activity. Phosphorylation at Serine 424 critically regulates this enzymatic activity. Research has demonstrated that Ser424 phosphorylation by casein kinase 2 (CK2) significantly increases HDAC3's deacetylase activity, while subsequent dephosphorylation by protein phosphatase 4 (PP4) decreases this activity. Mutation studies where Ser424 was substituted with alanine (S424A) showed severely reduced enzymatic activity, confirming that this phosphorylation site is absolutely critical for HDAC3 function. The phosphorylation does not appear to affect protein expression or subcellular localization but directly modulates catalytic activity .

What are the technical specifications of commercially available Phospho-HDAC3 (Ser424) antibodies?

Commercially available Phospho-HDAC3 (Ser424) antibodies are typically rabbit polyclonal antibodies that specifically recognize HDAC3 when phosphorylated at Serine 424. These antibodies demonstrate reactivity with human, mouse, and rat samples, with the molecular weight of the target protein being approximately 49 kDa. The antibodies are supplied in phosphate buffered saline (without Mg²⁺ and Ca²⁺), pH 7.4, 150mM NaCl, 0.02% sodium azide, and 50% glycerol, typically at a concentration of 1.0mg/mL. For optimal results, storage at -20°C or -80°C is recommended, and repeated freeze-thaw cycles should be avoided .

What are the recommended applications and dilutions for Phospho-HDAC3 (Ser424) antibody in laboratory research?

The Phospho-HDAC3 (Ser424) antibody can be utilized across multiple experimental applications with specific recommended dilutions:

The antibody has demonstrated sensitivity for detecting endogenous levels of phosphorylated HDAC3, making it suitable for studying physiological phosphorylation events without the need for overexpression systems .

How can researchers verify the specificity of phospho-HDAC3 (Ser424) antibody detection?

To verify antibody specificity, researchers should implement several validation approaches:

• Phosphatase Treatment Control: Treat one sample with lambda phosphatase before immunoblotting to confirm the signal is phosphorylation-dependent.

• Mutation Analysis: Compare detection between wild-type HDAC3 and S424A mutant HDAC3 in transfected cells. The antibody should show significantly reduced or no signal with the S424A mutant, as demonstrated in published research.

• Peptide Competition Assay: Pre-incubate the antibody with the phosphopeptide used as the immunogen (peptide sequence around phosphorylation site of Serine 424 (K-E-S(p)-D-V)) to block specific binding.

• Parallel Detection: Use both phospho-specific and total HDAC3 antibodies to confirm protein presence while separately evaluating phosphorylation status.

• Positive Controls: Include samples known to have high levels of HDAC3 Ser424 phosphorylation, such as cells treated with CK2 activators or PP4 inhibitors .

What experimental approaches can be used to modulate HDAC3 Ser424 phosphorylation in cellular systems?

Several experimental approaches can be employed to modulate HDAC3 Ser424 phosphorylation:

• CK2 Manipulation: Since casein kinase 2 (CK2) is the kinase responsible for phosphorylating HDAC3 at Ser424, researchers can:

  • Use CK2-specific inhibitors (such as TBB or CX-4945) to reduce phosphorylation

  • Overexpress CK2 to enhance phosphorylation

  • Employ siRNA against CK2 to reduce endogenous phosphorylation levels

• PP4 Manipulation: As protein phosphatase 4 (PP4) dephosphorylates HDAC3 at Ser424:

  • Overexpress PP4 catalytic subunit (PP4c) to decrease phosphorylation

  • Use siRNA knock-down of PP4c to increase phosphorylation

  • Analyze cells lacking PP4c to study the effects of prolonged phosphorylation

• HDAC3 Mutants: Create phosphomimetic (S424D or S424E) or phospho-null (S424A) HDAC3 mutants to study the functional consequences of constitutively phosphorylated or unphosphorylated states.

• N-terminal Deletion: Since the N-terminus of HDAC3 (residues 1-122) is necessary and sufficient for PP4c interaction, creating N-terminal deletion mutants can disrupt the HDAC3-PP4 interaction and consequently affect phosphorylation dynamics .

How does the HDAC3-PP4 interaction influence experimental design for studying Ser424 phosphorylation?

The HDAC3-PP4 interaction presents important considerations for experimental design. Research has shown that HDAC3 uniquely copurifies with both the catalytic (PP4c) and regulatory (PP4R1) subunits of the protein serine/threonine phosphatase 4 complex. This interaction is specific to HDAC3 and not observed with other class I HDACs like HDAC1 and HDAC2.

When designing experiments to study Ser424 phosphorylation, researchers should consider:

• Domain-Specific Interactions: The N-terminus of HDAC3 (residues 1-122) is both necessary and sufficient for HDAC3-PP4c interaction. Deletion analysis showed that C-terminal deletion mutants (1-180 and 1-122) bind PP4c, while N-terminal deletion mutants (122-428 and 180-428) do not. This domain specificity should inform the design of truncation mutants.

• Phosphatase Activity Controls: HDAC3 complexes display protein phosphatase activity, which may complicate phosphorylation analysis. Including phosphatase inhibitors when preparing cell extracts for phosphorylation studies is essential.

• Inverse Relationship: Multiple experimental approaches (overexpression, siRNA knock-down, and analysis of cells lacking PP4c) have demonstrated that HDAC3 activity is inversely proportional to PP4c abundance. This relationship should be considered when interpreting results from phosphorylation studies.

• Co-immunoprecipitation Specificity: When studying protein interactions, it's crucial to note that PP4c co-precipitates with anti-HDAC3 but not with preimmune sera, anti-HDAC1, or anti-HDAC2, highlighting the specificity of this interaction .

What are the most effective methods for quantifying changes in HDAC3 enzymatic activity in relation to Ser424 phosphorylation?

To effectively quantify changes in HDAC3 enzymatic activity in relation to Ser424 phosphorylation, researchers can employ the following methods:

• Histone Deacetylase Activity Assay: Immunoprecipitate HDAC3 (wild-type or mutants) from cell extracts using specific antibodies and measure its ability to deacetylate core histones. This can be quantified using:

  • Radioactively labeled acetylated histone substrates

  • Fluorometric HDAC activity assays using synthetic acetylated substrates

  • Mass spectrometry to measure changes in histone acetylation status

• Comparative Analysis of Phosphorylation States: Compare the deacetylase activity of:

  • Wild-type HDAC3 vs. S424A (phospho-null) mutant

  • Wild-type HDAC3 vs. S424D/E (phosphomimetic) mutant

  • HDAC3 from cells with manipulated CK2 or PP4 levels

• Correlation Analysis: Perform parallel assays to correlate:

  • Phosphorylation levels (measured by phospho-specific antibodies)

  • Deacetylase activity (measured by activity assays)

  • Interaction with co-repressors N-CoR and SMRT (measured by co-immunoprecipitation)

• In vitro Reconstitution: Purify recombinant HDAC3 and subject it to in vitro phosphorylation by CK2 or dephosphorylation by PP4, followed by enzymatic activity measurements to establish direct causality .

How do other post-translational modifications interact with Ser424 phosphorylation to regulate HDAC3 function?

While the search results focus primarily on Ser424 phosphorylation, a comprehensive approach to HDAC3 regulation requires consideration of multiple post-translational modifications and their potential interplay:

• Phosphorylation Crosstalk: Other phosphorylation sites on HDAC3 may interact with Ser424 phosphorylation. Though the search results mention Ser405 as having little effect on deacetylase activity, researchers should investigate whether prior phosphorylation at other sites primes or inhibits Ser424 phosphorylation.

• Acetylation-Phosphorylation Interplay: Since HDAC3 is a deacetylase that removes acetyl groups from proteins, researchers should examine whether HDAC3 itself undergoes acetylation that might influence its phosphorylation state or vice versa.

• Ubiquitination and Stability: Investigators should explore whether Ser424 phosphorylation affects HDAC3 stability by influencing its ubiquitination and subsequent proteasomal degradation.

• SUMOylation Effects: SUMO modification often affects protein localization and complex formation. Researchers might investigate whether SUMOylation of HDAC3 affects its interaction with PP4 or CK2, thereby indirectly influencing Ser424 phosphorylation.

• Redox Regulation: Oxidative stress can affect protein phosphorylation. Studies could examine whether cellular redox state influences HDAC3 phosphorylation at Ser424.

Experimental approaches should include mass spectrometry-based proteomics to identify the complete repertoire of HDAC3 modifications, followed by site-directed mutagenesis to create combinatorial mutants that can reveal modification interdependencies .

How does HDAC3 Ser424 phosphorylation status integrate with transcriptional repression pathways?

HDAC3's deacetylase activity is critical for its function in transcriptional repression, and this activity is directly regulated by Ser424 phosphorylation. HDAC3 deacetylase activity is stimulated by interactions with the N-CoR and SMRT co-repressor proteins. Together, these three proteins form a functional complex that represses transcription associated with nuclear hormone receptors and other transcription factors, including Rev-Erb, COUP-TF, DAX1, MAD, and Pit-1.

Researchers investigating the integration of Ser424 phosphorylation with transcriptional pathways should consider:

• Phosphorylation-Dependent Co-repressor Recruitment: Determine whether Ser424 phosphorylation affects HDAC3's ability to interact with N-CoR and SMRT co-repressors using co-immunoprecipitation experiments with wild-type, S424A, and S424D/E HDAC3 variants.

• Target Gene Expression Analysis: Perform RNA-seq or qRT-PCR on cells expressing wild-type HDAC3, S424A, or S424D/E mutants to identify genes whose expression is specifically affected by Ser424 phosphorylation status.

• Chromatin Immunoprecipitation (ChIP): Use ChIP assays to determine whether Ser424 phosphorylation affects HDAC3 recruitment to specific genomic loci or alters histone acetylation patterns at target genes.

• Nuclear Receptor Signaling: Given HDAC3's role in nuclear receptor-mediated repression, examine how hormonal signaling might influence Ser424 phosphorylation status and consequent transcriptional outcomes .

What experimental considerations are important when studying HDAC3 phosphorylation in different cellular compartments?

HDAC3 is described as both a nuclear and cytoplasmic protein, suggesting that its phosphorylation status may differ between cellular compartments and contribute to compartment-specific functions. When studying HDAC3 phosphorylation in different cellular locations, researchers should consider:

• Subcellular Fractionation: Perform careful subcellular fractionation to isolate nuclear, cytoplasmic, and possibly other compartments (mitochondria, endoplasmic reticulum) before analyzing HDAC3 phosphorylation status in each fraction.

• Compartment-Specific Distribution of Regulatory Enzymes: Determine the subcellular distribution of CK2 (the kinase) and PP4 (the phosphatase) to understand compartment-specific phosphorylation dynamics.

• Immunofluorescence Microscopy: Use dual staining with total HDAC3 and phospho-specific antibodies to visualize the spatial distribution of phosphorylated HDAC3 within cells. The recommended dilution for immunofluorescence is 1:200.

• Nuclear-Cytoplasmic Shuttling: Investigate whether phosphorylation at Ser424 affects HDAC3's nuclear import/export dynamics using techniques such as:

  • Leptomycin B treatment to block nuclear export

  • Photoactivatable or photoconvertible HDAC3 fusion proteins to track movement

  • FRAP (Fluorescence Recovery After Photobleaching) to measure mobility

• Stimulus-Dependent Relocalization: Examine whether cellular stimuli that activate CK2 or PP4 alter the subcellular distribution of phosphorylated HDAC3 .

How can researchers investigate the relationship between HDAC3 Ser424 phosphorylation and disease mechanisms?

Given HDAC3's involvement in epigenetic regulation and transcriptional repression, its dysregulation through altered phosphorylation may contribute to various disease states. Researchers investigating disease connections should:

• Clinical Sample Analysis: Examine phospho-HDAC3 levels in patient-derived samples using the phospho-specific antibody at the recommended dilution of 1:200 for immunohistochemistry of paraffin-embedded sections. Compare levels between healthy and diseased tissues.

• Disease Model Systems: Utilize relevant disease models (cancer cell lines, neurodegenerative disease models, inflammatory models) to study how disease-associated stimuli affect HDAC3 phosphorylation and consequent activity.

• Pharmacological Interventions: Test how HDAC inhibitors, particularly HDAC3-selective inhibitors, affect the phosphorylation status of HDAC3 and whether their efficacy depends on the phosphorylation state.

• Genetic Association Studies: Investigate whether single nucleotide polymorphisms (SNPs) near the Ser424 site are associated with disease risk or progression.

• Therapeutic Target Validation: Determine whether modulating Ser424 phosphorylation (via CK2 inhibitors or PP4 regulators) might have therapeutic potential in diseases where HDAC3 activity is implicated.

• Phosphorylation-Dependent Protein Interactions: Identify disease-relevant proteins that interact specifically with phosphorylated or non-phosphorylated HDAC3 using phospho-specific protein interaction proteomics .

What are common technical challenges when detecting phosphorylated HDAC3 and how can they be addressed?

Detecting phosphorylated proteins presents several technical challenges. For Phospho-HDAC3 (Ser424) specifically, researchers should be aware of:

• Phosphatase Activity During Sample Preparation:

  • Challenge: Endogenous phosphatases can dephosphorylate HDAC3 during sample preparation.

  • Solution: Add phosphatase inhibitors (sodium orthovanadate, sodium fluoride, β-glycerophosphate) to lysis buffers. Consider using commercially available phosphatase inhibitor cocktails.

• Antibody Specificity:

  • Challenge: Cross-reactivity with unphosphorylated HDAC3 or other phosphoproteins.

  • Solution: Include appropriate controls (phosphatase-treated samples, S424A mutant) and validate with peptide competition assays.

• Low Signal Intensity:

  • Challenge: Low abundance of phosphorylated HDAC3 in certain cell types or conditions.

  • Solution: Enrich for phosphoproteins using phospho-enrichment techniques (TiO₂ beads, IMAC) before immunoblotting.

• Basal Phosphorylation Levels:

  • Challenge: High basal phosphorylation making experimental manipulations difficult to detect.

  • Solution: Serum-starve cells before treatment to reduce basal phosphorylation.

• Antibody Storage and Handling:

  • Challenge: Antibody degradation affecting detection sensitivity.

  • Solution: Store at -20°C or -80°C, avoid repeated freeze-thaw cycles, and aliquot the antibody upon receipt .

How can researchers optimize conditions for studying the dynamic regulation of HDAC3 phosphorylation?

To effectively study the dynamic regulation of HDAC3 phosphorylation, researchers should consider:

• Time-Course Experiments:

  • Design experiments with multiple time points after stimulation or inhibition to capture the kinetics of phosphorylation changes.

  • Include both short (minutes) and long (hours) time points to distinguish between immediate and delayed effects.

• Dose-Response Relationships:

  • Titrate concentrations of CK2 activators/inhibitors or PP4 modulators to determine threshold effects.

  • Plot dose-response curves for both phosphorylation and deacetylase activity to identify potential disconnects.

• Physiological Stimuli:

  • Identify physiological stimuli that modulate HDAC3 phosphorylation (growth factors, hormones, stress conditions).

  • Compare phosphorylation kinetics across different stimuli to identify common regulatory principles.

• Pulse-Chase Analysis:

  • Use phosphate-free media supplemented with radioactive orthophosphate (³²P) to label newly phosphorylated proteins.

  • Chase with non-radioactive phosphate to determine the half-life of the phosphorylated state.

• Single-Cell Techniques:

  • Employ phospho-flow cytometry or immunofluorescence microscopy to assess cell-to-cell variability in phosphorylation responses.

  • Consider FRET-based biosensors to monitor HDAC3 phosphorylation in living cells .

What control experiments are essential when using phospho-HDAC3 (Ser424) antibody in complex biological systems?

When using phospho-HDAC3 (Ser424) antibody in complex biological systems like tissue samples or primary cells, essential control experiments include:

• Antibody Validation Controls:

  • Phosphatase Treatment: Treat duplicate samples with lambda phosphatase to confirm signal specificity.

  • Peptide Competition: Pre-incubate antibody with phosphopeptide immunogen to block specific binding.

  • Knockout/Knockdown Validation: Use HDAC3 knockout or knockdown samples to confirm antibody specificity.

• Sample-Specific Controls:

  • Total HDAC3 Detection: Always run parallel blots or sequential probing for total HDAC3 to normalize phospho-signal.

  • Loading Controls: Include appropriate loading controls (β-actin, GAPDH for total lysates; histone H3, lamin for nuclear fractions).

  • Positive Controls: Include samples known to have high phospho-HDAC3 levels (e.g., cells treated with CK2 activators).

• Experimental Design Controls:

  • Untreated/Vehicle Controls: Include proper untreated samples or vehicle controls for all treatments.

  • Time-Matched Controls: For time-course experiments, include time-matched controls to account for temporal variations in basal phosphorylation.

  • Biological Replicates: Perform experiments with biological replicates (different donors, animals, or independent cell cultures) to ensure reproducibility.

• Technical Validation:

  • Multiple Detection Methods: Validate key findings using alternative detection methods (e.g., mass spectrometry-based phosphoproteomics).

  • Alternative Antibodies: If available, use antibodies from different sources or raised against different epitopes containing phospho-Ser424 .

What emerging techniques could enhance our understanding of HDAC3 Ser424 phosphorylation dynamics?

Several cutting-edge techniques could significantly advance our understanding of HDAC3 Ser424 phosphorylation:

• CRISPR-Based Approaches:

  • CRISPR knock-in of fluorescent tags to endogenous HDAC3 for live-cell imaging

  • CRISPR base editing to introduce the S424A mutation at the endogenous locus

  • CRISPRa/CRISPRi systems to modulate expression of CK2 or PP4 with temporal precision

• Advanced Microscopy Techniques:

  • Super-resolution microscopy to visualize nanoscale distribution of phosphorylated HDAC3

  • FRET/FLIM-based biosensors to monitor HDAC3 phosphorylation in real-time

  • Single-molecule tracking to analyze how phosphorylation affects HDAC3 mobility and interactions

• Proteomics Approaches:

  • Proximity labeling (BioID, APEX) to identify phosphorylation-dependent interactors

  • Crosslinking mass spectrometry to map structural changes induced by phosphorylation

  • Targeted proteomics (PRM/MRM) for absolute quantification of phosphorylation stoichiometry

• Structural Biology:

  • Cryo-EM studies of HDAC3 complexes in different phosphorylation states

  • Hydrogen-deuterium exchange mass spectrometry to probe conformational changes

  • AlphaFold2 or RoseTTAFold prediction of structural differences between phosphorylated and non-phosphorylated states

• Systems Biology:

  • Multi-omics integration combining phosphoproteomics, transcriptomics, and epigenomics

  • Mathematical modeling of HDAC3 phosphorylation/dephosphorylation cycles

  • Network analysis to position HDAC3 phosphorylation within broader signaling networks .

How can researchers investigate the evolutionary significance of HDAC3 Ser424 phosphorylation?

To investigate the evolutionary significance of HDAC3 Ser424 phosphorylation, researchers could pursue several approaches:

• Comparative Genomics:

  • Analyze the conservation of Ser424 and surrounding sequence across species, from simple organisms to complex vertebrates

  • Identify when this phosphorylation site emerged during evolution

  • Determine whether conservation correlates with specific organismal features or developmental complexity

• Phylogenetic Analysis:

  • Construct phylogenetic trees of HDAC3 from diverse species

  • Map the presence/absence of the Ser424 site onto these trees

  • Identify evolutionary transitions where this site emerged or was lost

• Functional Conservation Studies:

  • Express HDAC3 from different species in mammalian cells and assess phosphorylation

  • Test whether phosphorylation-dependent regulation is conserved across species

  • Identify species-specific differences in the regulation or consequences of phosphorylation

• Evolutionary Rate Analysis:

  • Calculate evolutionary rates (dN/dS) around the Ser424 site

  • Determine whether this site is under purifying selection (conserved function) or positive selection (adaptive evolution)

  • Compare evolutionary rates with other phosphorylation sites on HDAC3

• Ancestral Sequence Reconstruction:

  • Reconstruct ancestral HDAC3 sequences at key evolutionary nodes

  • Express reconstructed proteins and test their phosphorylation properties

  • Determine when phosphorylation-dependent regulation first appeared .

What are promising approaches for translating basic knowledge about HDAC3 phosphorylation into therapeutic applications?

Translating knowledge about HDAC3 Ser424 phosphorylation into therapeutic applications could follow several promising avenues:

• Targeted Drug Development:

  • Design small molecules that specifically inhibit HDAC3 in a phosphorylation-dependent manner

  • Develop peptide mimetics that disrupt interactions between phosphorylated HDAC3 and its specific binding partners

  • Create bifunctional degraders (PROTACs) that selectively target phosphorylated HDAC3 for degradation

• Biomarker Development:

  • Validate phospho-HDAC3 as a biomarker for disease diagnosis or prognosis

  • Develop clinical-grade phospho-HDAC3 detection methods for patient stratification

  • Create companion diagnostics to identify patients likely to respond to HDAC3-targeting therapies

• Combination Therapy Approaches:

  • Test combinations of CK2 inhibitors with HDAC inhibitors for synergistic effects

  • Identify downstream effectors of phosphorylated HDAC3 as alternative drug targets

  • Develop rational drug combinations based on phosphorylation-dependent vulnerabilities

• Precision Medicine Strategies:

  • Characterize patient-specific alterations in HDAC3 phosphorylation pathways

  • Design personalized treatment approaches based on phosphorylation status

  • Develop phosphorylation-based predictive models for treatment response

• Novel Delivery Systems:

  • Design nanoparticle-based delivery systems to target drugs to tissues with aberrant HDAC3 phosphorylation

  • Develop cell-penetrating peptides that modulate HDAC3 phosphorylation in specific cellular compartments

  • Create spatiotemporally controlled drug release systems to precisely modulate HDAC3 phosphorylation dynamics .