Phospho-HDAC8 (Ser39) Antibody

What is HDAC8 and why is its phosphorylation at Serine 39 biologically significant?

HDAC8 (Histone deacetylase 8) catalyzes the deacetylation of lysine residues on the N-terminal part of core histones (H2A, H2B, H3, and H4). This deacetylation process provides a tag for epigenetic repression and plays crucial roles in:

• Transcriptional regulation

• Cell cycle progression

• Developmental events

• Smooth muscle cell contractility

• Chromatin structure modification

Phosphorylation of HDAC8 at Serine 39 (Ser39) by cyclic AMP-dependent protein kinase A (PKA) functions as a negative regulatory mechanism. When phosphorylated at this site, HDAC8's deacetylase activity is substantially reduced, particularly toward histones H3 and H4 . This post-translational modification represents a key mechanism by which cells can rapidly modulate epigenetic regulation in response to cellular signaling events.

Research has demonstrated that PKA phosphorylates HDAC8 exclusively on serine residues, with Ser39 being the principal phosphoacceptor site. Forskolin treatment, which activates adenyl cyclase and increases cAMP levels, enhances HDAC8 phosphorylation, while PKA inhibitors such as H-89 block this phosphorylation .

How can I determine the optimal working concentration for Phospho-HDAC8 (Ser39) antibodies in different applications?

The optimal working concentration varies by application and must be empirically determined. Here are evidence-based recommendations based on manufacturer guidelines:

To optimize antibody concentration:

• Perform a dilution series experiment

• Include appropriate positive controls (cells treated with forskolin to activate PKA)

• Include negative controls (cells treated with PKA inhibitors like H-89)

• For Western blotting, include HDAC8(S39A) mutant samples as specificity controls if possible

The antibody demonstrates reactivity with human, mouse, and rat samples, making it versatile for cross-species research applications .

What experimental controls should be included when using Phospho-HDAC8 (Ser39) antibodies?

Proper experimental controls are essential for interpreting results obtained with phospho-specific antibodies:

Positive Controls:

• Cells or tissues treated with forskolin (0.5-10 μM for 30-45 minutes), which activates PKA and increases HDAC8 Ser39 phosphorylation

• Recombinant GST-HDAC8 phosphorylated in vitro by purified PKA

Negative Controls:

• Cells pre-treated with PKA inhibitors (H-89 at 5-10 μM or PKI at 10-20 μM)

• HDAC8 knockout or knockdown cells

• Cells expressing HDAC8(S39A) mutant, which cannot be phosphorylated at position 39

Specificity Controls:

• Peptide competition assay using the immunizing phosphopeptide

• Dual detection with total HDAC8 antibody on parallel blots or through stripping and reprobing

• Phosphatase treatment of cell lysates to remove phosphorylation

These controls help verify that the observed signal truly represents phosphorylated HDAC8 rather than non-specific binding or cross-reactivity with other phosphoproteins.

What are the best methods for studying the dynamics of HDAC8 phosphorylation in living cells?

To investigate the temporal dynamics of HDAC8 phosphorylation:

• Time-course experiments:

  • Treat cells with forskolin (5-10 μM) for varying durations (5, 15, 30, 45, 60, 120 minutes)

  • Harvest cells and analyze by Western blotting with Phospho-HDAC8 (Ser39) antibody

  • Plot the phosphorylation intensity normalized to total HDAC8 versus time

• Pulse-chase experiments with radiolabeled phosphate:

  • Incubate cells with 32P-orthophosphate (0.5 mCi/ml for 4 hours)

  • Add PKA activators/inhibitors at different time points

  • Immunoprecipitate HDAC8 and analyze by SDS-PAGE and autoradiography

• Phospho-HDAC8 (Ser39) cell-based ELISA:

  • Use colorimetric cell-based ELISA kits specific for Phospho-HDAC8 (Ser39)

  • Plate cells directly in 96-well format

  • Apply treatments and quantify phosphorylation in a high-throughput manner

• Live-cell imaging:

  • Express HDAC8 fused to a fluorescent protein

  • Use phospho-specific antibodies conjugated to different fluorophores in fixed cells at different timepoints

  • Analyze subcellular localization changes upon phosphorylation

This multifaceted approach enables robust characterization of the kinetics and cellular context of HDAC8 phosphorylation events.

How does HDAC8 phosphorylation at Ser39 affect its enzymatic activity and substrate specificity?

Phosphorylation of HDAC8 at Ser39 significantly impacts its enzymatic function:

Substrate Preference Shifts:

Research indicates that phosphorylation alters HDAC8 substrate specificity:

The molecular basis for this selectivity involves:

• Altered enzyme conformation affecting the substrate binding pocket

• Potential changes in protein-protein interactions with cofactors

• Modified subcellular localization (nuclear vs. cytoplasmic distribution)

Researchers can leverage phospho-mimetic mutants (S39E) and phospho-resistant mutants (S39A) to further dissect these mechanisms in experimental systems .

What are the critical parameters for optimizing immunohistochemistry protocols with Phospho-HDAC8 (Ser39) antibodies?

Optimizing immunohistochemistry (IHC) with phospho-specific antibodies requires careful attention to several parameters:

Tissue Preparation:

• Use freshly prepared tissue sections when possible

• For paraffin-embedded tissues, ensure rapid fixation (<24 hours in 10% neutral buffered formalin)

• Consider using phosphatase inhibitors (sodium fluoride, sodium orthovanadate) in fixatives to preserve phosphorylation status

Antigen Retrieval:

• Heat-induced epitope retrieval in citrate buffer (pH 6.0) is recommended

• Alternative: Tris-EDTA buffer (pH 9.0) if signal is weak

• Optimize retrieval time (15-30 minutes) and temperature

Blocking and Antibody Incubation:

• Block with 5-10% normal serum from the species of the secondary antibody

• Include 1% BSA to reduce non-specific binding

• Primary antibody dilution: Start at 1:100-1:300

• Incubate at 4°C overnight for optimal sensitivity

Signal Detection:

• For DAB detection systems, optimize development time to prevent overdevelopment

• For fluorescent detection, select fluorophores with minimal spectral overlap if performing multiplex staining

• Consider tyramide signal amplification for detecting low-abundance phosphoproteins

Critical Controls:

• Adjacent tissue sections treated with lambda phosphatase

• Tissue from animals/cells treated with PKA activators and inhibitors

• Peptide competition controls to verify specificity

This methodical approach ensures reliable detection of phosphorylated HDAC8 within the complex environment of tissue specimens.

What is the relationship between HDAC8 phosphorylation and subcellular localization?

The phosphorylation status of HDAC8 at Ser39 influences its subcellular distribution, with important implications for its function:

Normal Localization Pattern:

HDAC8 is typically found in both nuclear and cytoplasmic compartments, with exclusion from nucleoli. In cells showing smooth muscle differentiation, HDAC8 exhibits pronounced cytoplasmic localization .

Effects of Phosphorylation:

Research indicates that PKA-mediated phosphorylation at Ser39 can alter this distribution pattern:

• Phosphorylated HDAC8 may show increased cytoplasmic retention

• The nuclear-cytoplasmic shuttling rate may be affected

• Association with specific subcellular structures (chromatin, cytoskeleton) can change

Experimental Approaches to Study Localization:

To investigate these dynamics, researchers can employ:

• Immunofluorescence with phospho-specific antibodies (recommended dilution 1:50-200)

• Subcellular fractionation followed by Western blotting

• Live-cell imaging of fluorescently tagged HDAC8 variants (wild-type, S39A, S39E)

• Co-localization studies with nuclear envelope markers, chromatin markers, or cytoskeletal components

Functional Implications:

The altered localization upon phosphorylation contributes to HDAC8's reduced activity on nuclear histone substrates and may redirect its activity toward cytoplasmic substrates, representing a sophisticated regulatory mechanism beyond simple inhibition of catalytic activity.

How can I troubleshoot weak or absent signals when using Phospho-HDAC8 (Ser39) antibodies?

When encountering weak or absent signals with Phospho-HDAC8 (Ser39) antibodies, systematically address these potential issues:

Sample Preparation Issues:

• Phosphorylation loss during sample processing:

  • Add phosphatase inhibitors (50 mM NaF, 1 mM Na3VO4, 10 mM β-glycerophosphate) to all buffers

  • Keep samples cold throughout processing

  • Use fresh samples; avoid repeated freeze-thaw cycles

• Insufficient PKA activation:

  • Verify forskolin activity with a known PKA substrate

  • Optimize forskolin concentration (5-10 μM) and treatment time (30-45 minutes)

  • Consider alternative PKA activators (8-Br-cAMP, dibutyryl-cAMP)

Antibody-Related Issues:

• Antibody concentration:

  • Try different dilutions; for Western blot, test 1:500, 1:1000, and 1:2000

  • For immunostaining applications, use more concentrated antibody (1:50-1:100)

• Detection system sensitivity:

  • Switch to more sensitive detection (e.g., from colorimetric to chemiluminescent)

  • Try signal amplification systems (e.g., biotin-streptavidin)

  • Use higher-sensitivity substrates for Western blot detection

• Cross-reactivity concerns:

  • Verify antibody lot performance with positive control lysates

  • Perform peptide competition assays to confirm specificity

Protocol Optimization:

• Western blot transfer issues:

  • Optimize transfer conditions for proteins in the HDAC8 size range (~42 kDa)

  • Consider semi-dry vs. wet transfer methods

  • Verify transfer with reversible protein staining

• Antigen retrieval for tissue sections:

  • Test different antigen retrieval methods (heat, enzymatic)

  • Extend antigen retrieval time for heavily fixed samples

This systematic troubleshooting approach addresses the most common reasons for weak phospho-specific antibody signals.

How can phosphorylation of HDAC8 at Ser39 affect its interactions with protein complexes?

HDAC8 phosphorylation at Ser39 modulates its ability to participate in multi-protein complexes, with significant functional consequences:

Altered Protein-Protein Interactions:

Phosphorylation introduces a negatively charged phosphate group that can:

• Disrupt existing protein-protein interactions

• Create new binding sites for phospho-binding domain-containing proteins

• Induce conformational changes affecting interaction surfaces

Specific Complex Alterations:

Research indicates several impacts on HDAC8's participation in protein complexes:

Experimental Approaches to Study Complex Formation:

To investigate these dynamics:

• Co-immunoprecipitation:

  • Immunoprecipitate with anti-HDAC8 or anti-phospho-HDAC8 antibodies

  • Compare binding partners between phosphorylated and non-phosphorylated states

  • Use phospho-mimetic (S39E) and phospho-resistant (S39A) mutants

• Proximity labeling:

  • Fuse HDAC8 variants to BioID or APEX2

  • Identify differential interactors when HDAC8 is phosphorylated vs. non-phosphorylated

• Crosslinking mass spectrometry:

  • Compare crosslinked complexes from cells with activated or inhibited PKA

  • Identify structural changes in complexes containing phosphorylated HDAC8

These methodological approaches reveal how phosphorylation serves as a molecular switch regulating HDAC8's participation in diverse cellular processes.

What are the best approaches for quantifying changes in HDAC8 phosphorylation levels?

Accurate quantification of HDAC8 phosphorylation requires careful selection of methods and rigorous normalization:

Western Blot Quantification:

• Dual detection approach:

  • Probe replicate blots with phospho-specific and total HDAC8 antibodies

  • Calculate phospho-HDAC8/total HDAC8 ratio

  • Use appropriate loading controls (GAPDH, β-actin)

• Quantification parameters:

  • Use chemiluminescence detection within the linear range

  • Capture multiple exposure times to ensure linearity

  • Use densitometry software (ImageJ, Image Studio Lite) for analysis

ELISA-Based Quantification:

Phospho-HDAC8 (Ser39) colorimetric cell-based ELISA kits provide quantitative data:

• Plate cells directly in 96-well format

• Use dual detection of phospho-HDAC8 and total HDAC8

• Calculate normalized phosphorylation ratio

Mass Spectrometry Approaches:

For absolute quantification:

• Selected reaction monitoring (SRM):

  • Use isotopically labeled phosphopeptide standards

  • Monitor specific transitions for phosphorylated and non-phosphorylated HDAC8 peptides

  • Calculate stoichiometry of phosphorylation

• Parallel reaction monitoring (PRM):

  • Targeted MS method with higher specificity

  • Identify and quantify phosphopeptides containing Ser39

  • Compare peak areas to synthetic standards

In-Cell Quantification:

For spatial information:

• Quantitative immunofluorescence with phospho-specific antibodies

• Normalize signal to total HDAC8 staining

• Analyze nuclear/cytoplasmic distribution ratio

These complementary approaches provide robust quantification of HDAC8 phosphorylation status under diverse experimental conditions.

How does HDAC8 phosphorylation at Ser39 affect chromatin remodeling and gene expression?

The phosphorylation of HDAC8 at Ser39 has profound effects on chromatin structure and gene expression patterns:

Direct Effects on Histone Deacetylation:

When HDAC8 is phosphorylated at Ser39 by PKA, its deacetylase activity toward histone substrates is significantly reduced . This leads to:

• Increased histone acetylation at HDAC8 target sites

• More open chromatin structure at affected genomic regions

• Enhanced accessibility for transcription factors

Genome-Wide Effects:

Studies indicate that HDAC8 phosphorylation affects specific gene sets:

Experimental Approaches to Study Transcriptional Effects:

To investigate these effects comprehensively:

• ChIP-seq analysis:

  • Compare histone acetylation patterns in cells with wild-type vs. phosphomimetic HDAC8

  • Identify genomic regions with differential HDAC8 occupancy upon PKA activation

  • Correlate with gene expression changes

• RNA-seq:

  • Profile transcriptome changes upon forskolin treatment

  • Compare with HDAC8 inhibitor treatment

  • Identify genes specifically regulated by HDAC8 phosphorylation

• ATAC-seq:

  • Analyze chromatin accessibility changes linked to HDAC8 phosphorylation

  • Map open chromatin regions in PKA-activated vs. control cells

This multilayered approach provides insight into how HDAC8 phosphorylation contributes to signal-responsive gene regulation through chromatin-based mechanisms.

What considerations are important when validating the specificity of Phospho-HDAC8 (Ser39) antibodies?

Thorough validation of phospho-specific antibodies is critical for research integrity and involves several complementary approaches:

Essential Validation Experiments:

• Peptide competition assays:

  • Pre-incubate antibody with phosphorylated and non-phosphorylated peptides

  • A specific signal should be blocked only by the phospho-peptide

  • Use graduated concentrations to determine blocking efficiency

• Phosphatase treatment controls:

  • Treat one portion of positive control lysate with lambda phosphatase

  • Compare Western blot signals between treated and untreated samples

  • Phospho-specific signal should disappear after phosphatase treatment

• Genetic validation:

  • Test antibody reactivity in HDAC8 knockout/knockdown cells

  • Compare wild-type HDAC8 with S39A mutant expression

  • Phospho-specific signal should be absent in S39A mutant samples

• PKA modulation:

  • Treat cells with forskolin to enhance phosphorylation

  • Treat cells with H-89 or PKI to inhibit phosphorylation

  • Verify signal response mirrors expected phosphorylation changes

Advanced Validation Approaches:

• Mass spectrometry correlation:

  • Quantify Ser39 phosphorylation by MS

  • Compare with antibody-based quantification

  • Establish correlation between methods

• Orthogonal antibody comparison:

  • Test multiple phospho-specific antibodies from different vendors

  • Compare detection patterns across applications

  • Consistent results increase confidence in specificity

These rigorous validation steps ensure that experimental observations genuinely reflect HDAC8 phosphorylation status rather than artifacts or cross-reactivity.

How can I design experiments to investigate the relationship between HDAC8 phosphorylation and cellular physiology?

Designing experiments to link HDAC8 phosphorylation to cellular functions requires a multifaceted approach:

Genetic Models:

• HDAC8 phospho-mutant expression:

  • Generate stable cell lines expressing:

    • Wild-type HDAC8

    • Phospho-resistant HDAC8 (S39A)

    • Phospho-mimetic HDAC8 (S39E)

  • Compare phenotypes under normal and stress conditions

• CRISPR-Cas9 genome editing:

  • Create cell lines with endogenous HDAC8-S39A knock-in

  • Compare with wild-type cells following PKA activation

Pharmacological Approaches:

• PKA pathway modulation:

  • Activators: Forskolin (5-10 μM), 8-Br-cAMP, dibutyryl-cAMP

  • Inhibitors: H-89 (5-10 μM), PKI (10-20 μM)

  • Time-course and dose-response experiments

• Combined HDAC and PKA inhibition:

  • Compare HDAC8-specific inhibitors alone vs. with PKA modulators

  • Assess additive or synergistic effects

Functional Assays:

Systems Biology Integration:

• Multi-omics approach:

  • Integrate phosphoproteomics, acetylomics, and transcriptomics

  • Map signaling networks connecting HDAC8 phosphorylation to cellular responses

  • Identify pathway crosstalk mechanisms

• Temporal dynamics analysis:

  • Time-resolved measurements following PKA activation

  • Pathway reconstruction based on kinetic parameters

  • Mathematical modeling of HDAC8 phosphorylation/dephosphorylation cycle

This comprehensive experimental design strategy enables robust investigation of HDAC8 phosphorylation's role in cellular physiology across multiple scales.

What are the technical considerations for phospho-proteomic analysis of HDAC8 phosphorylation?

Mass spectrometry-based phosphoproteomic analysis of HDAC8 requires specific technical considerations to ensure accurate identification and quantification:

Sample Preparation:

• Enrichment strategies:

  • Immunoprecipitation with HDAC8-specific antibodies

  • Phosphopeptide enrichment methods (TiO2, IMAC, phospho-tyrosine antibodies)

  • Consider fractionation to reduce sample complexity

• Preservation of phosphorylation:

  • Add phosphatase inhibitors to all buffers:

    • Sodium fluoride (50 mM)

    • Sodium orthovanadate (1 mM)

    • β-glycerophosphate (10 mM)

  • Maintain cold temperatures throughout processing

  • Rapid protein extraction and denaturation

MS Analysis Parameters:

• Peptide coverage considerations:

  • Multiple proteases beyond trypsin (Lys-C, Glu-C) to generate diverse peptides

  • The Ser39-containing peptide is within residues 5-54 of human HDAC8

  • Ensure detection of peptides containing Ser39 site

• Fragmentation methods:

  • HCD provides good coverage of phosphopeptides

  • ECD/ETD preserves labile phosphorylation during fragmentation

  • Consider combined fragmentation approaches

• Detection strategies:

  • Targeted methods (PRM/SRM) for known phosphosites

  • Data-dependent acquisition for discovery

  • Data-independent acquisition for comprehensive coverage

Quantification Approaches:

• Label-based quantification:

  • SILAC for cell culture experiments

  • TMT/iTRAQ for multiplexed analysis

  • Heavy-labeled synthetic phosphopeptide standards

• Label-free quantification:

  • Spectral counting

  • MS1 intensity-based methods

  • MS2 fragment-based methods

Bioinformatic Analysis:

• Database search parameters:

  • Include phosphorylation (+79.9663 Da) as variable modification

  • Consider other potential PTMs (acetylation, ubiquitination)

  • Implement site localization algorithms (Ascore, ptmRS)

• Validation criteria:

  • Manual verification of MS/MS spectra for Ser39 phosphopeptides

  • Require diagnostic ions confirming phosphorylation at Ser39 vs. nearby sites

  • Apply stringent FDR thresholds for phosphosite identification

These technical considerations ensure reliable phosphoproteomic analysis of HDAC8 phosphorylation at Ser39 and other potentially relevant sites.

How can I integrate Phospho-HDAC8 (Ser39) research into broader epigenetic signaling studies?

Integrating HDAC8 phosphorylation research into comprehensive epigenetic studies requires connecting this specific modification to broader regulatory networks:

Multi-Level Regulatory Analysis:

• Signaling pathway integration:

  • Map PKA-HDAC8 axis within cAMP signaling networks

  • Identify crosstalk with other pathways affecting histone modifications

  • Analyze temporal coordination between phosphorylation and acetylation changes

• Epigenetic crosstalk mapping:

  • Investigate how HDAC8 phosphorylation affects other epigenetic marks:

    • Histone acetylation at specific lysines (H3K9ac, H3K27ac)

    • DNA methylation patterns

    • Chromatin remodeling complex activity

Systems Approach Implementation:

• Integrated multi-omics:

  • Combine data from:

    • Phosphoproteomics (HDAC8 and other components)

    • Acetylomics (histone and non-histone targets)

    • Transcriptomics (gene expression changes)

    • Chromatin accessibility (ATAC-seq, DNase-seq)

• Network modeling:

  • Construct directed graphs connecting:

    • PKA activation → HDAC8 phosphorylation

    • HDAC8 phosphorylation → histone acetylation changes

    • Histone acetylation → transcription factor binding

    • Transcription factor binding → gene expression

Physiological Context Expansion:

Translational Research Connections:

• Therapeutic targeting:

  • Investigate compounds that modulate HDAC8 phosphorylation

  • Compare with direct HDAC8 inhibitors

  • Analyze combination approaches targeting both activity and phosphorylation

• Biomarker development:

  • Evaluate Phospho-HDAC8 (Ser39) as a potential biomarker using validated antibodies

  • Correlate with disease progression or treatment response

  • Develop tissue-specific detection protocols

This integrated approach positions HDAC8 phosphorylation research within the broader context of epigenetic regulation and signaling networks, enhancing its biological significance and translational potential.