Phospho-HDAC2 (Ser394) Antibody

What is the Phospho-HDAC2 (Ser394) Antibody and what are its basic applications?

Phospho-HDAC2 (Ser394) Antibody is a rabbit polyclonal antibody specifically designed to detect HDAC2 (Histone deacetylase 2) protein only when phosphorylated at serine 394. This antibody has demonstrated reactivity with human, mouse, and rat samples, making it versatile for cross-species research applications. The primary applications include Western Blot (WB) and immunofluorescence/immunocytochemistry (IF/ICC), with some variants also suitable for ELISA-based detection systems . The antibody is particularly valuable for researchers investigating epigenetic regulation mechanisms, as HDAC2 plays a crucial role in histone deacetylation processes that influence gene expression patterns.

What is the significance of HDAC2 phosphorylation at Ser394 in cellular functions?

Phosphorylation of HDAC2 at Ser394 represents a critical post-translational modification that significantly alters the protein's functional properties. Research has demonstrated that this specific phosphorylation event is associated with reduced deacetylase activity of HDAC2 . The modification at Ser394 has paradoxical effects - while decreasing HDAC2's enzymatic activity, it simultaneously enhances HDAC2's capacity to form co-repressor complexes, interact with transcription factors, recruit CREB-binding protein (CBP), and undergo acetylation on lysine residues . These combined effects ultimately lead to increased transrepression activity, suggesting that phosphorylation at Ser394 serves as a regulatory switch that repurposes HDAC2 function rather than simply activating or deactivating the protein.

How do I properly store and handle Phospho-HDAC2 (Ser394) Antibody for optimal results?

For optimal antibody performance and longevity, store Phospho-HDAC2 (Ser394) Antibody at -20°C for up to one year from the receipt date. The antibody is typically formulated in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide to maintain stability . Avoid repeated freeze-thaw cycles as they can degrade antibody quality and compromise experimental results. When working with the antibody, maintain cold chain conditions and aliquot the stock solution into smaller volumes for single-use applications. Before each experiment, briefly centrifuge the antibody vial to collect the solution at the bottom of the tube. For Western blot applications, dilution ranges typically fall between 1:500-1:2000, while ELISA applications may require more dilute preparations (approximately 1:20000) . Always optimize the antibody concentration for your specific experimental conditions and cell types.

What are the recommended protocols for detecting phosphorylated HDAC2 (Ser394) in cell culture experiments?

To effectively detect phosphorylated HDAC2 at Ser394 in cell culture experiments, researchers should implement a systematic approach combining cell preparation, treatment, and detection methods. Begin by seeding cells (e.g., H292 or similar cell lines) at a density of 3.0-5.0 × 10^5 cells in 6-well culture plates containing appropriate medium (such as RPMI-1640 with 10% FBS without antibiotics) and allow attachment overnight . For experiments investigating factors affecting HDAC2 phosphorylation, treat cells with relevant stimuli - for example, cigarette smoke extract (CSE) at 2.5% for specific time intervals (0.5h, 2h, 6h) has been used to induce phosphorylation changes .

For protein extraction, lyse cells in buffer containing phosphatase inhibitors (typically 0.5% NP-40, 25 mM Tris, 100 mM NaCl, pH 7.4) for 30 minutes on ice . Separate proteins by SDS-PAGE and transfer to PVDF membranes for immunoblotting with Phospho-HDAC2 (Ser394) antibody (typical dilution 1:500-1:2000) . For increased specificity, consider enriching phosphorylated HDAC2 through immunoprecipitation before Western blotting. For quantitative assessment of phosphorylation levels, colorimetric cell-based ELISA kits specific for Phospho-HDAC2 (Ser394) can provide high sensitivity detection across a range of >5000 cells .

How can I effectively use transfection methods to study HDAC2 phosphorylation at Ser394?

For transfection-based studies of HDAC2 phosphorylation at Ser394, prepare cells at appropriate density (4 × 10^6 cells in 100 mm dishes containing 7 ml of RPMI-1640 with 10% FBS without antibiotics) and allow overnight attachment . The following day, transiently transfect cells with expression vectors containing wild-type HDAC2 (1-488), or mutant constructs such as S394A (where serine is replaced with alanine to prevent phosphorylation) or multiple mutants like S394/422/424A .

For optimal transfection efficiency, use Lipofectamine 2000 according to the manufacturer's protocol with 4-8 μg of plasmid DNA. For co-transfection experiments (e.g., with CBP or CBP mutants lacking HAT activity), ensure equal total DNA amounts across all conditions by supplementing with empty vector plasmids . After 24 hours of transfection, treat cells with relevant stimuli to induce phosphorylation. For subsequent analysis, perform immunoprecipitation using anti-flag antibodies if flag-tagged HDAC2 constructs were used, followed by Western blotting with phospho-specific antibodies to detect the phosphorylation status at Ser394.

For knockdown studies to investigate kinases involved in HDAC2 phosphorylation, transfect cells with 100 pmol of relevant siRNAs (e.g., CK2α siRNA) for 24 hours before introducing HDAC2 expression constructs . This sequential transfection approach helps establish the relationship between specific kinases and HDAC2 phosphorylation at Ser394.

What controls should be included when studying HDAC2 phosphorylation at Ser394?

When studying HDAC2 phosphorylation at Ser394, a comprehensive set of controls is essential for result validation. Include the following controls:

• Positive and negative phosphorylation controls: Treat cells with known phosphorylation inducers like okadaic acid (100 nM, 1h) as a positive control . Untreated cells serve as negative controls.

• Antibody specificity controls: Include samples from cells expressing HDAC2 mutants where Ser394 is replaced with alanine (S394A) to confirm antibody specificity for the phosphorylated residue .

• Total HDAC2 detection: Always probe for total HDAC2 levels alongside phospho-specific detection to normalize phosphorylation signals and account for expression level variations.

• Pathway validation controls: When studying the kinases responsible for Ser394 phosphorylation, include controls where the suspected kinase (CK2α) is inhibited pharmaceutically or through siRNA-mediated knockdown .

• Multiple mutant controls: Compare single S394A mutants with multiple mutants (S394/422/424A) to distinguish the specific contribution of Ser394 phosphorylation from other phosphorylation events .

• Transfection controls: For transfection experiments, include empty vector controls and GFP expression constructs to monitor transfection efficiency and control for non-specific effects of the transfection procedure .

How does protein kinase CK2-mediated phosphorylation of HDAC2 at Ser394 regulate its deacetylase activity?

Protein kinase CK2-mediated phosphorylation of HDAC2 at Ser394 establishes a sophisticated regulatory mechanism that inversely correlates with deacetylase activity. Research has demonstrated that this phosphorylation event significantly reduces HDAC2's enzymatic activity toward histone substrates . The mechanism involves direct interaction between HDAC2 and serine-phosphorylated protein kinase CK2α, suggesting a specific molecular recognition process rather than random kinase-substrate interactions .

Paradoxically, while phosphorylation diminishes HDAC2's deacetylase function, it simultaneously enhances the protein's capacity to form co-repressor complexes and interact with transcription factors. This phosphorylation facilitates HDAC2 recruitment of CREB-binding protein (CBP), which subsequently catalyzes acetylation of HDAC2 on lysine residues . This phospho-acetylation dual modification creates a functionally distinct version of HDAC2 with increased transrepression activity despite reduced catalytic function.

To effectively study this regulatory mechanism, researchers should implement CK2α knockdown experiments using specific siRNAs, followed by assessment of HDAC2 phosphorylation status at Ser394 and concurrent measurement of deacetylase activity. Additionally, comparative studies between wild-type HDAC2 and phospho-deficient mutants (S394A) can elucidate the direct relationship between this specific phosphorylation event and enzymatic activity.

What is the relationship between HDAC2 Ser394 phosphorylation and inflammatory conditions?

HDAC2 phosphorylation at Ser394 plays a significant role in inflammatory conditions, particularly in steroid resistance observed in chronic inflammatory diseases . Research has established that phospho-acetylation of HDAC2 (phosphorylation at Ser394 followed by acetylation on lysine residues) negatively regulates its deacetylase activity, contributing to the molecular mechanisms underlying steroid resistance .

In inflammatory contexts, oxidative stress appears to be a key mediator affecting HDAC2 phosphorylation. Studies using cigarette smoke extract (CSE) as an oxidative stress inducer have demonstrated altered HDAC2 phosphorylation patterns, suggesting a direct link between redox status and HDAC2 function . This relationship is particularly relevant in respiratory inflammatory conditions like chronic obstructive pulmonary disease (COPD), where oxidative stress is prevalent.

To investigate this relationship, researchers should design experiments examining HDAC2 phosphorylation states in inflammatory versus normal conditions, using appropriate cellular models (such as bronchial epithelial cell lines for respiratory inflammation). Analysis should include assessment of:

• Phosphorylation status at Ser394 under inflammatory stimuli

• Correlation between phosphorylation levels and deacetylase activity

• Impact of anti-inflammatory interventions on HDAC2 phosphorylation

• Functional consequences of preventing Ser394 phosphorylation in inflammatory response models

How can phosphatase activity modulation be used to study HDAC2 Ser394 phosphorylation dynamics?

Modulating phosphatase activity provides a powerful approach to investigate HDAC2 Ser394 phosphorylation dynamics. Since phosphorylation status represents an equilibrium between kinase and phosphatase activities, manipulating phosphatases offers unique insights into regulatory mechanisms. To implement this approach, researchers can use phosphatase inhibitors like okadaic acid (100 nM) to prevent dephosphorylation and study the natural phosphorylation rate at Ser394 .

For comprehensive analysis of phosphatase involvement, prepare cell lysates in phosphatase storage buffer (50 mM Tris HCl pH 8, 150 mM NaCl, 1% NP40, 0.1% SDS, 0.5% deoxycholate, without phosphatase inhibitors) . Measure phosphatase activity using commercial serine/threonine phosphatase assay kits with phosphopeptide substrates like RRA(pT)VA. To isolate specific phosphatase contributions, remove endogenous phosphate by applying samples to Sephadex G-25 resin columns and centrifuging at 600 × g for 5 minutes at 4°C .

Compare phosphatase activity levels with HDAC2 Ser394 phosphorylation status under various treatment conditions to establish correlation patterns. Additionally, investigate the specific role of PP2A (Protein Phosphatase 2A), which has been implicated in regulating phosphorylation of nuclear proteins including HDACs. This approach helps construct a comprehensive understanding of the dynamic regulation of HDAC2 phosphorylation in different cellular contexts.

What are common challenges in detecting phosphorylated HDAC2 at Ser394 and how can they be addressed?

Detecting phosphorylated HDAC2 at Ser394 presents several technical challenges that researchers should anticipate and address methodically. Common issues include:

• Low signal intensity: Phosphorylation events are often transient and affect only a fraction of the total protein pool. To enhance detection, optimize cell lysis conditions using buffers containing phosphatase inhibitors (sodium fluoride, sodium orthovanadate, β-glycerophosphate) . Consider phospho-protein enrichment techniques before Western blotting, and use high-sensitivity detection systems such as chemiluminescent substrates with extended exposure times.

• High background signals: Non-specific antibody binding can obscure phospho-specific signals. Improve specificity by optimizing antibody dilutions (typically 1:500-1:2000 for Western blot) , extending blocking steps (5% BSA in TBST is often more effective than milk for phospho-epitopes), and implementing more stringent washing procedures (additional washes with higher salt concentrations).

• Phosphorylation instability: HDAC2 Ser394 phosphorylation can be rapidly lost during sample preparation due to endogenous phosphatase activity. Prevent this by processing samples quickly at 4°C and including phosphatase inhibitor cocktails in all buffers. For highly sensitive applications, consider adding okadaic acid (100 nM) to samples immediately upon collection .

• Antibody cross-reactivity: Phospho-specific antibodies may recognize similar phosphorylated epitopes in other proteins. Validate specificity using phospho-deficient HDAC2 mutants (S394A) as negative controls . Additionally, competing phosphorylation at nearby residues (such as S422 and S424) might affect antibody recognition; use multiple antibody sources or detection methods to confirm results.

How can I distinguish between specific HDAC2 Ser394 phosphorylation and other post-translational modifications?

Distinguishing specific HDAC2 Ser394 phosphorylation from other post-translational modifications requires a multi-faceted approach combining genetic, biochemical, and analytical techniques:

• Site-directed mutagenesis: Generate phospho-deficient HDAC2 mutants where Ser394 is replaced with alanine (S394A) to prevent phosphorylation specifically at this site . Compare these with wild-type HDAC2 and other phospho-site mutants (S422A, S424A) to isolate Ser394-specific effects. Additionally, create phospho-mimetic mutants (S394D or S394E) to simulate constitutive phosphorylation.

• Phosphatase treatment: Treat immunoprecipitated HDAC2 with lambda phosphatase to remove all phosphorylation modifications, then compare with untreated samples to determine total phosphorylation contribution. Use specific inhibitors to selectively block certain phosphatases (e.g., okadaic acid for PP2A inhibition) .

• Mass spectrometry analysis: Implement targeted phospho-proteomics to precisely map all phosphorylation sites on HDAC2. This approach can quantitatively assess the relative abundance of phosphorylation at Ser394 versus other sites under different experimental conditions.

• Sequential immunoprecipitation: First immunoprecipitate total HDAC2, then perform a second immunoprecipitation with Phospho-HDAC2 (Ser394) antibody to enrich specifically for this phospho-form. Western blotting can then be performed to detect other modifications (acetylation, ubiquitination) that may co-occur with Ser394 phosphorylation.

• Kinase manipulation: Selectively inhibit or deplete CK2α using specific inhibitors or siRNA approaches, as this kinase has been identified as responsible for Ser394 phosphorylation . This approach helps distinguish CK2-mediated phosphorylation at Ser394 from modifications catalyzed by other kinases.

What factors influence the reproducibility of HDAC2 Ser394 phosphorylation experiments?

Several factors critically influence the reproducibility of HDAC2 Ser394 phosphorylation experiments, requiring careful experimental design and standardization:

• Cell culture conditions: Growth factors in serum can activate signaling pathways affecting phosphorylation. Standardize serum concentrations and consider serum starvation (6-12 hours) before treatments to establish consistent baseline phosphorylation levels. Cell density and passage number also significantly impact phosphorylation patterns; maintain consistent plating densities (3.0-5.0 × 10^5 cells in 6-well plates) and use cells within a limited passage range .

• Treatment timing and conditions: HDAC2 phosphorylation is dynamic and time-dependent. Establish detailed time courses for treatments (e.g., 0.5h, 2h, 6h for CSE exposure) to capture peak phosphorylation events . Environmental variables like temperature, pH, and CO2 levels should be strictly controlled during treatments.

• Lysis conditions: Phosphorylation status can change rapidly during sample processing. Standardize cell lysis protocols by using consistent buffer compositions (0.5% NP-40, 25 mM Tris, 100 mM NaCl with protease and phosphatase inhibitors, pH 7.4), lysis durations (30 minutes on ice), and sample handling procedures . Process all experimental conditions simultaneously to minimize variation.

• Antibody quality and batch effects: Antibody performance can vary between lots. When possible, complete experimental series using the same antibody lot, and include internal standard samples across blots for normalization. Validate each new antibody lot against known positive controls.

• Protein load and transfer efficiency: Inconsistent protein loading or variable transfer efficiency can confound phosphorylation analysis. Implement rigorous protein quantification before loading, use consistent protein amounts (typically 20-50 μg for Western blots), and include loading controls (both total HDAC2 and housekeeping proteins) for normalization .

How does HDAC2 Ser394 phosphorylation interact with other post-translational modifications to create a regulatory code?

HDAC2 Ser394 phosphorylation operates within a complex network of post-translational modifications that collectively form a regulatory code determining HDAC2 function. Research indicates that phosphorylation at Ser394 facilitates subsequent acetylation on lysine residues, establishing a phospho-acetylation sequence that fundamentally alters HDAC2 activity . This sequential modification pattern suggests a hierarchical regulatory system where phosphorylation serves as a priming event for additional modifications.

Beyond acetylation, S-nitrosylation represents another significant modification affecting HDAC2 function. Research has identified cysteine residues (Cys-262 and Cys-274) as targets for S-nitrosylation, which can induce HDAC2 release from chromatin in neurons . This raises interesting questions about potential crosstalk between phosphorylation at Ser394 and S-nitrosylation events - whether they operate independently, synergistically, or antagonistically.

To investigate these complex interactions, researchers should implement comprehensive modification mapping using mass spectrometry techniques coupled with site-specific mutant studies. Sequential treatment protocols can help establish modification hierarchies, while proximity ligation assays might identify modification-specific protein interaction partners. This research direction promises to reveal how combinatorial modifications create functionally distinct HDAC2 populations within cells, potentially explaining context-specific functions in different tissues and disease states.

What is the relationship between HDAC2 Ser394 phosphorylation and neurodegenerative disorders?

The relationship between HDAC2 Ser394 phosphorylation and neurodegenerative disorders represents an emerging area of investigation with significant therapeutic implications. HDAC2 has been implicated in neurodegenerative conditions through its regulation of neuronal gene expression and memory formation processes . Phosphorylation at Ser394, which modulates HDAC2's deacetylase activity and interactions with transcription factors, may serve as a critical regulatory mechanism in neuronal function and dysfunction.

In neuronal contexts, HDAC2 post-translational modifications appear to have specialized roles. For example, S-nitrosylation of HDAC2 in neurons induces its release from chromatin without affecting enzyme activity, subsequently increasing acetylation of histones surrounding neurotrophin-dependent gene promoters . This raises the possibility that phosphorylation at Ser394 might interact with neuronal-specific modification patterns to regulate HDAC2 function in brain tissues.

To explore this relationship, researchers should:

• Compare HDAC2 Ser394 phosphorylation patterns in brain tissues from neurodegenerative disease models versus controls

• Examine how neuronal stimulation affects HDAC2 phosphorylation

• Investigate potential crosstalk between Ser394 phosphorylation and neuronal-specific modifications like S-nitrosylation

• Assess how modulating HDAC2 phosphorylation affects neuronal gene expression patterns, particularly genes implicated in neurodegeneration

How can phosphorylation-specific antibodies like Phospho-HDAC2 (Ser394) be integrated with advanced imaging techniques for in situ analysis?

Integrating phosphorylation-specific antibodies like Phospho-HDAC2 (Ser394) with advanced imaging techniques offers powerful approaches for in situ analysis of phosphorylation dynamics at cellular and subcellular levels. This integration requires careful optimization of protocols to maintain phospho-epitope integrity while achieving high-resolution visualization.

For super-resolution microscopy applications, researchers should optimize fixation protocols specifically for phospho-epitope preservation. Paraformaldehyde fixation (4%, 10-15 minutes) followed by methanol post-fixation (-20°C, 10 minutes) often yields superior results for phosphorylated nuclear proteins. When implementing immunofluorescence detection, use higher antibody concentrations than for Western blotting (typically starting at 1:200-1:500 dilutions) and extend incubation times (overnight at 4°C) .

To achieve multiplexed detection of different HDAC2 modifications simultaneously, consider sequential immunostaining protocols with careful antibody stripping between rounds, or implement spectral unmixing approaches with fluorophores having distinct emission profiles. For live-cell imaging of phosphorylation dynamics, phospho-specific intrabodies or FRET-based biosensors could be developed, though these would require extensive validation against the established Phospho-HDAC2 (Ser394) antibodies.

The combination of phospho-specific immunodetection with proximity ligation assays (PLA) offers particularly powerful insights, enabling visualization of specific interaction partners of phosphorylated HDAC2 within intact cells. This approach could reveal spatial and temporal patterns of phosphorylation-dependent interactions that are lost in biochemical assays, providing unprecedented insights into the functional consequences of HDAC2 Ser394 phosphorylation in different cellular compartments and physiological contexts.