Phospho-HDAC5 (Ser498) Antibody

What is HDAC5 and what is its general function in cells?

HDAC5 (Histone deacetylase 5) is responsible for the deacetylation of lysine residues on the N-terminal part of core histones (H2A, H2B, H3, and H4). This deacetylation creates a tag for epigenetic repression and plays important roles in transcriptional regulation, cell cycle progression, and developmental events . HDAC5 belongs to the class IIa histone deacetylase family and functions through the formation of large multiprotein complexes. It is particularly involved in muscle maturation by repressing transcription of myocyte enhancer MEF2C and in the MTA1-mediated epigenetic regulation of ESR1 expression in breast cancer . Additionally, HDAC5 serves as a corepressor of RARA (retinoic acid receptor alpha) and plays a role in the repression of microRNA-10a, thereby influencing inflammatory responses .

What is the significance of HDAC5 phosphorylation at Ser498?

Phosphorylation of HDAC5 at Ser498 is a critical regulatory mechanism that controls HDAC5 subcellular localization and function. When phosphorylated at Ser498 (along with Ser259), HDAC5 binds to 14-3-3 proteins, which promotes its export from the nucleus to the cytoplasm . This nuclear export is a key regulatory step that relieves HDAC5-mediated repression of target genes, allowing for their activation . The phosphorylation state of Ser498 is therefore a molecular switch that determines whether HDAC5 can repress transcription in the nucleus or is sequestered in the cytoplasm, permitting gene expression .

What are the recommended applications and dilutions for Phospho-HDAC5 (Ser498) antibody?

Phospho-HDAC5 (Ser498) antibodies are validated for multiple applications with specific recommended dilution ranges:

These recommendations serve as starting points and may require optimization for specific experimental systems . When designing experiments, it is advisable to include appropriate positive control samples where HDAC5 phosphorylation is known to be induced, such as cells treated with PKC activators like PMA .

How should Phospho-HDAC5 (Ser498) antibody be stored and handled to maintain optimal activity?

For maximum stability and performance of Phospho-HDAC5 (Ser498) antibody, follow these storage and handling guidelines:

• Store the antibody at -20°C for long-term storage (up to one year from the date of receipt) .

• For frequent use and short-term storage (up to one month), keep at 4°C to avoid repeated freeze-thaw cycles .

• The antibody is typically supplied in liquid form in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide .

• When aliquoting, use sterile tubes and minimize freeze-thaw cycles, as each cycle can decrease antibody activity by approximately 10% .

• Prior to use, gently mix the antibody solution by inversion rather than vortexing to prevent protein denaturation .

Proper storage and handling are essential for maintaining antibody specificity and sensitivity, particularly for phospho-specific antibodies that detect post-translational modifications .

What controls should be included when using Phospho-HDAC5 (Ser498) antibody?

To ensure the reliability and interpretability of results, include the following controls when using Phospho-HDAC5 (Ser498) antibody:

• Positive Control: Use cell lysates known to contain phosphorylated HDAC5, such as:

  • VEC cells (vascular endothelial cells)

  • Jurkat cells

  • Cells treated with PMA (phorbol 12-myristate 13-acetate) to activate PKC

  • Endothelial cells exposed to fluid shear stress

• Negative Controls:

  • Samples treated with phosphatase to remove phosphorylation

  • Use of phospho-blocking peptide specific to the Ser498 epitope

  • Use of non-phosphorylated recombinant HDAC5 protein

• Specificity Controls:

  • Compare with total HDAC5 antibody to evaluate the proportion of phosphorylated protein

  • Use HDAC5 phospho-mutants (S498A) where the serine is replaced with alanine

• Technical Controls:

  • Secondary antibody only control to check for non-specific binding

  • Loading controls (β-actin, GAPDH) for Western blot normalization

Validation data from the literature shows that immunohistochemistry analysis of human breast carcinoma using HDAC5 (Phospho-Ser498) antibody can be effectively blocked with the phospho-peptide, demonstrating specificity . Similarly, Western blot analysis of Jurkat cell lysates shows specific bands that can be blocked with the phospho-peptide .

How does the interplay between different HDAC5 phosphorylation sites affect its function?

The function of HDAC5 is regulated by a complex interplay between multiple phosphorylation sites, with different functional outcomes:

• Dual Phosphorylation at Ser259 and Ser498:

  • Both sites serve as 14-3-3 protein binding sites

  • Mutation of either single site has minimal effect on 14-3-3 binding

  • Simultaneous mutation of both sites (S259/498A) completely abolishes 14-3-3 binding

  • Both sites are phosphorylated in response to CaMK activation, leading to nuclear export

• Phosphorylation at Ser280 (by PKA):

  • Counteracts the effects of Ser259/498 phosphorylation

  • Prevents the nuclear export of HDAC5 even when Ser259/498 are phosphorylated

  • Interferes with the interaction between HDAC5 and 14-3-3 proteins

  • Changes the conformation of HDAC5 to block 14-3-3 binding

• Other Phosphorylation Sites:

  • Ser611 in the activation domain (AD) shows prominent phosphorylation (10-50%)

  • Ser1108 in the nuclear export signal (NES) shows high phosphorylation levels (25-80%)

  • These sites modulate protein-protein interactions, particularly with transcription factors and components of the NCoR complex

Research has shown that mutation of phosphorylation sites within the activation domain increases association with HDAC3 and MEF2D, while phosphorylation sites in the nuclear export signal are critical for protein interactions required for transcriptional repression .

What is the role of HDAC5 Ser498 phosphorylation in fluid shear stress responses in endothelial cells?

Fluid shear stress plays a crucial role in vascular homeostasis, and HDAC5 phosphorylation at Ser498 is a key mediator in this process:

• Mechanistic Pathway:

  • Fluid shear stress stimulates HDAC5 phosphorylation at Ser259/498 in endothelial cells through a calcium/calmodulin-dependent pathway

  • This phosphorylation triggers nuclear export of HDAC5

  • Nuclear export of HDAC5 leads to:

    • Dissociation of HDAC5 from myocyte enhancer factor-2 (MEF2)

    • Enhanced MEF2 transcriptional activity

    • Increased expression of KLF2 (Krüppel-like factor 2) and eNOS (endothelial nitric oxide synthase)

• Functional Consequences:

  • KLF2 and eNOS are key mediators of flow anti-inflammatory and anti-atherosclerotic actions

  • HDAC5 phosphorylation and nuclear export contribute to:

    • Protection of blood vessels from atherosclerosis

    • Enhancement of endothelial cell survival

    • Regulation of vascular tone

    • Anti-inflammatory effects

• Experimental Evidence:

  • Adenoviral overexpression of a phosphorylation-defective HDAC5 mutant (S259A/S498A) renders HDAC5 resistant to flow-induced nuclear export

  • This mutant suppresses:

    • Flow-mediated MEF2 transcriptional activity

    • Expression of KLF2 and eNOS

    • Flow-inhibitory effect on monocyte adhesion to endothelial cells

These findings suggest that HDAC5 phosphorylation at Ser498 is a critical regulatory mechanism in vascular endothelial cells, with potential implications for therapeutic approaches targeting atherosclerosis .

How can phospho-mutants of HDAC5 be used to study its function?

Phospho-mutants of HDAC5 are powerful tools for dissecting the specific roles of different phosphorylation sites:

• Types of HDAC5 Phospho-mutants:

  Mutant	Description	Functional Effect	Research Applications
  S498A	Serine to alanine at position 498	Prevents phosphorylation at this site	Study site-specific effects
  S259A	Serine to alanine at position 259	Prevents phosphorylation at this site	Study site-specific effects
  S259/498A	Double mutation of both serines	Completely blocks 14-3-3 binding and nuclear export	Study coordinated phosphorylation
  S280A	Serine to alanine at position 280	Prevents PKA-mediated phosphorylation	Study PKA pathway interactions
  S280D	Serine to aspartic acid at position 280	Mimics phosphorylation	Study constitutive effects of phosphorylation

• Experimental Approaches:

  • Transfection or viral transduction of phospho-mutants in cell culture

  • Creation of transgenic animal models expressing phospho-mutants

  • Comparison of nuclear vs. cytoplasmic localization using immunofluorescence or cell fractionation

  • Analysis of protein-protein interactions using co-immunoprecipitation

  • Measurement of target gene expression changes

• Research Examples:

  • The S259/498A double mutant was used to demonstrate the requirement of both sites for 14-3-3 binding and nuclear export

  • The S280D phosphomimetic mutant was shown to be resistant to nuclear exclusion in response to PMA, mimicking the effect of PKA activation

  • In endothelial cells, the S259/498A mutant was used to show that HDAC5 phosphorylation mediates flow-induced KLF2 and eNOS expression

• Methodological Considerations:

  • When designing phospho-mutants, consider using multiple approaches (phospho-null and phosphomimetic)

  • Validate mutant expression levels to ensure they match endogenous protein

  • Confirm that mutations don't disrupt other functions or protein folding

  • Use appropriate controls, including wild-type HDAC5 and empty vectors

Why might I observe different results between Western blot and immunocytochemistry when using Phospho-HDAC5 (Ser498) antibody?

Discrepancies between Western blot and immunocytochemistry results can arise from several factors:

• Sample Preparation Differences:

  • Western blot typically involves denaturing conditions that may expose epitopes differently than fixed cells in immunocytochemistry

  • Phosphorylation can be lost during sample processing if phosphatase inhibitors are inadequate

• Dynamic Phosphorylation:

  • HDAC5 phosphorylation is dynamic and can vary widely (10-80% depending on the site)

  • Cell fixation captures a moment in time, while lysate preparation may allow changes in phosphorylation state

• Subcellular Localization:

  • Phosphorylated HDAC5 shuttles between nucleus and cytoplasm

  • Immunocytochemistry reveals this spatial information, while Western blot shows total levels

  • Nuclear export following phosphorylation may make the signal appear weaker in nuclear fractions

• Methodological Solutions:

  • Use phosphatase inhibitors consistently in all sample preparations

  • Perform subcellular fractionation when preparing Western blot samples

  • Include time-course experiments to capture the dynamic nature of phosphorylation

  • Use dual immunofluorescence with total HDAC5 antibody for colocalization studies

Research has shown that phosphorylation at Ser498 may be differentially regulated across cell types and in response to various stimuli, which can contribute to experimental variability .

How can I distinguish between the effects of HDAC5 phosphorylation at Ser498 versus other sites?

Distinguishing between different phosphorylation sites on HDAC5 requires specific experimental approaches:

• Use of Site-Specific Antibodies:

  • Compare results using antibodies specific to different phosphorylation sites (e.g., pSer259, pSer498, pSer280)

  • Verify antibody specificity using phospho-peptide blocking experiments

  • Use antibodies that recognize multi-phosphorylated forms (e.g., pSer632/pSer661/pSer486)

• Phospho-Mutant Approach:

  • Generate and compare single mutants (S498A, S259A, S280A) and combination mutants (S259/498A)

  • Assess differential effects on:

    • 14-3-3 binding

    • Nuclear export

    • Target gene expression

    • Interaction with partner proteins

• Kinase Manipulation:

  • Use specific kinase activators or inhibitors that preferentially target different sites

  • For example:

    • PKA activators (forskolin, cAMP analogs) primarily affect Ser280

    • PMA affects Ser259/498 through PKC/PKD pathways

    • CaMK activators target Ser259/498

• Mass Spectrometry Approaches:

  • Use targeted MS/MS approaches with parallel reaction monitoring (PRM) to quantify site-specific phosphorylation

  • This allows precise measurement of phosphorylation levels at individual sites

  • Can calculate ratios of phosphorylated to unphosphorylated peptides

Research has demonstrated that while single mutations at either Ser259 or Ser498 had minimal effect on CaMK-inducible 14-3-3 binding, the double mutation completely abolished this interaction, highlighting the importance of studying both individual and combined phosphorylation events .

What factors might affect the specificity of Phospho-HDAC5 (Ser498) antibody?

Several factors can influence the specificity of Phospho-HDAC5 (Ser498) antibody:

• Sequence Homology Between Class IIa HDACs:

  • The region around Ser498 in HDAC5 shares homology with other class IIa HDACs

  • Some antibodies may cross-react with phosphorylated forms of HDAC4 and HDAC7

  • For example, the phospho-HDAC4 (Ser632)/HDAC5 (Ser661)/HDAC7 (Ser486) antibody may also recognize HDAC5 phosphorylated at Ser498

• Post-Translational Modifications:

  • HDAC5 undergoes multiple post-translational modifications beyond phosphorylation, including ubiquitination

  • These modifications may alter epitope recognition

• Antibody Production Variables:

  • Different commercial antibodies use various immunogen sequences around the Ser498 site

  • Some use peptides spanning amino acids 464-513 , while others may use different regions

  • Polyclonal antibodies contain a mixture of antibodies that may recognize different epitopes

• Experimental Validation Methods:

  • Use phospho-blocking peptides to confirm specificity

  • Test antibody reactivity with phospho-mutant proteins (S498A)

  • Test on samples treated with phosphatases

  • Use immunodepletion with total HDAC5 antibody to confirm identity

• Cross-reactivity:

  • Some anti-phospho-HDAC5 (Ser498) antibodies may recognize unidentified proteins at different molecular weights

  • Validate specificity by comparing Western blot patterns with expected HDAC5 molecular weight (approximately 122 kDa)

When selecting an antibody, review the validation data and experimental approach used by the manufacturer to ensure it meets the requirements of your specific application and experimental system .

How can I design experiments to study the temporal dynamics of HDAC5 phosphorylation and nuclear export?

Studying the temporal dynamics of HDAC5 phosphorylation and nuclear export requires sophisticated experimental design:

• Live Cell Imaging Approaches:

  • Generate GFP-tagged HDAC5 constructs for real-time visualization

  • Use fluorescence recovery after photobleaching (FRAP) to measure nuclear-cytoplasmic shuttling rates

  • Implement time-lapse microscopy following stimulation with agents that induce phosphorylation

• Biochemical Time-Course Experiments:

  • Collect samples at multiple time points after stimulation (e.g., PMA, fluid shear stress)

  • Perform Western blots with phospho-specific antibodies

  • Combine with subcellular fractionation to track nuclear vs. cytoplasmic distribution

  • Quantify the kinetics of phosphorylation and nuclear export

• Advanced Proteomic Approaches:

  • Use Stable Isotope Labeling with Amino acids in Cell culture (SILAC) to quantify temporal changes

  • Implement pulse-chase experiments to track newly phosphorylated HDAC5

  • Apply targeted mass spectrometry to measure site-specific phosphorylation rates

• Kinase Activity Correlation:

  • Simultaneously monitor the activation of relevant kinases (CaMK, PKD, PKA)

  • Correlate kinase activity with HDAC5 phosphorylation and localization

  • Use specific kinase inhibitors to establish temporal sequence of events

• Dual Reporter Systems:

  • Combine GFP-tagged HDAC5 with RFP-tagged 14-3-3 proteins

  • Monitor their interaction and localization in real-time

  • Use fluorescence resonance energy transfer (FRET) to detect direct binding

• Experimental Example Design:

  • Establish baseline HDAC5 localization in serum-starved cells

  • Add stimulus (e.g., PMA, fluid shear stress) and collect samples at intervals (0, 5, 15, 30, 60, 120 min)

  • Process parallel samples for immunofluorescence and Western blot

  • Quantify nuclear/cytoplasmic ratio and phosphorylation levels at each time point

  • Correlate with downstream gene expression changes (e.g., MEF2 target genes)