Phospho-HIST1H3A (T6) Antibody

What is the biological significance of Histone H3 phosphorylation at Threonine 6?

Histone H3 phosphorylation at Threonine 6 (pH3T6) plays crucial roles in multiple cellular processes. This post-translational modification is involved in:

• Chromatin remodeling and accessibility

• Cell cycle regulation, particularly during mitosis

• Transcriptional activation and repression

• DNA damage response mechanisms

• Epigenetic regulation of gene expression

Research indicates that phosphorylation of Histone H3 at Thr6 has been linked to cancer progression, with high levels of pH3T6 correlating with poor prognosis in glioblastoma multiforme (GBM) . The phosphorylation is typically mediated by protein kinase C (PKC), and studies have shown that high pH3T6 levels correlate with increased PKC activity .

How should researchers validate the specificity of Phospho-Histone H3 (Thr6) antibodies?

Validating antibody specificity is critical for accurate interpretation of experimental results. For Phospho-Histone H3 (Thr6) antibodies, implement these methods:

• Peptide arrays: Quantify detection of the target histone modification while evaluating cross-reactivity with other histone modifications . This approach allows systematic testing against a panel of related phosphorylation sites and other modifications.

• Western blot with controls: Use samples with altered phosphorylation states:

  • Phosphatase-treated samples (negative control)

  • Samples treated with PKC activators (positive control)

  • Kinase inhibitor treatments (e.g., Enzastaurin for PKC inhibition)

• Immunoprecipitation followed by mass spectrometry: Verify that the immunoprecipitated material contains H3T6ph.

• Knockout/knockdown validation: Use cells with targeted disruption of kinases responsible for T6 phosphorylation.

• SNAP-ChIP methodology: Similar to approaches used for other histone modifications , where DNA-barcoded recombinant nucleosomes can validate specificity in chromatin immunoprecipitation assays.

For Western Blot:

• Extract histones using acid extraction method to enrich for histones

• Run 15-20 μg of histone extract on 15-18% SDS-PAGE

• Transfer to PVDF membrane (preferred over nitrocellulose for small proteins)

• Block with 5% BSA in TBST

• Incubate with primary antibody at recommended dilution (typically 1:1000-1:8000)

• Expected molecular weight: 15-18 kDa

For Immunofluorescence/Immunocytochemistry:

• Fix cells with 4% paraformaldehyde for 10 minutes

• Permeabilize with 0.1% Triton X-100 for 5 minutes

• Block with 1% BSA for 30 minutes

• Incubate with primary antibody overnight at 4°C

• Use a fluorescently labeled secondary antibody

• Co-stain with DAPI to visualize nuclei

For Tissue Sections:

• Use antigen retrieval with TE buffer pH 9.0 or citrate buffer pH 6.0

• Block endogenous peroxidase activity if using HRP detection

• Incubate with primary antibody (1:500-1:2000)

• Co-staining with cell-type specific antibodies can help identify affected cell populations

How does pH3T6 correlate with clinical outcomes in cancer studies?

Research on glioblastoma multiforme (GBM) has revealed significant correlations between pH3T6 levels and patient outcomes:

This data from a collection of 42 GBM patients indicates that pH3T6 is a signature associated with poor prognosis in patients treated with temozolomide and irradiation (TMZ+Irad treatment) . The study demonstrated that high pH3T6 levels correlated with high PKC activity, suggesting potential therapeutic targeting of this pathway.

What experimental approaches can distinguish between cell cycle-associated and transcription-associated pH3T6?

Histone H3 phosphorylation can occur during both mitosis and transcriptional activation. To distinguish between these functions:

• Cell cycle synchronization:

  • Use double thymidine block or serum starvation to synchronize cells

  • Collect samples at different cell cycle phases

  • Compare pH3T6 patterns across cell cycle

• Co-immunostaining with cell cycle markers:

  • pH3T6 + cyclin B1 (G2/M marker)

  • pH3T6 + Ki-67 (proliferation marker)

  • pH3T6 + PCNA (S phase marker)

• Transcription inhibition experiments:

  • Treat cells with α-amanitin or actinomycin D

  • Assess if pH3T6 levels decrease independently of cell cycle changes

• ChIP-seq analysis:

  • Compare pH3T6 enrichment with active transcription markers (H3K4me3, RNA Pol II)

  • Assess correlation with transcriptional start sites

This approach is similar to methods used for other phosphorylated histone residues, where researchers have shown that specific phosphorylation events like H3S10 can have distinct functions during interphase versus mitosis .

How do researchers isolate high-quality nuclear extracts for pH3T6 analysis?

For optimal detection of histone phosphorylation, nuclear extraction quality is critical:

• Acid extraction protocol for enriched histone preparation:

  • Isolate nuclei using hypotonic buffer with 0.1% NP-40

  • Extract histones with 0.2N HCl for 30 minutes on ice

  • Neutralize with 1M Tris-HCl pH 8.0

  • Concentrate using TCA precipitation or dialysis

• Phosphatase inhibitor considerations:

  • Always include sodium fluoride (10 mM)

  • Add sodium orthovanadate (1 mM)

  • Include β-glycerophosphate (10 mM)

  • Keep samples cold throughout extraction

• Quantification and storage:

  • Determine protein concentration using Bradford assay

  • Store at -80°C in aliquots to avoid freeze-thaw cycles

  • Add 50% glycerol for long-term storage

This approach preserves phosphorylation states that are often labile during standard protein extraction procedures.

What challenges exist in interpreting pH3T6 cross-reactivity with other histone modifications?

Interpreting phospho-histone antibody reactivity presents several challenges:

• Proximity to other modification sites:

  • T6 is adjacent to K4 and K9, both major methylation sites

  • Research shows potential cross-talk between H3T6 phosphorylation and H3K4 methylation

  • Consider using antibodies that specifically recognize combinatorial modifications (e.g., H3K4me3/T6ph)

• Antibody validation methods comparison:

  Validation Method	Advantages	Limitations
  Peptide arrays	High-throughput, quantitative	Limited to linear epitopes
  Western blot	Detects full-length protein	Limited spatial information
  Immunofluorescence	Provides spatial context	Potential fixation artifacts
  Specificity testing with modified nucleosomes	Physiological substrate	More complex to implement

• Technical considerations:

  • Some antibodies show cross-reactivity with other phosphorylated residues

  • Ensure validation in multiple applications

  • Consider confirming key findings with at least two independent antibody clones

Researchers should carefully assess antibody validation data from manufacturers and independent studies to understand potential limitations.

How can researchers quantify pH3T6 levels across different experimental conditions?

Several approaches allow for accurate quantification of phosphorylation levels:

• Western blot densitometry:

  • Normalize pH3T6 signal to total H3 levels

  • Use at least three biological replicates

  • Include positive controls (e.g., PKC activator-treated cells)

  • Present data as fold-change relative to control

• Quantitative immunofluorescence:

  • Measure nuclear pH3T6 fluorescence intensity

  • Normalize to DAPI or total H3 staining

  • Use automated image analysis software for unbiased quantification

  • Analyze >100 cells per condition

• Flow cytometry:

  • Perform intracellular staining for pH3T6

  • Dual staining with DNA content marker allows cell cycle correlation

  • Quantify percentage of positive cells and mean fluorescence intensity

  • Suitable for rare cell populations

• ChIP-qPCR:

  • Quantify pH3T6 enrichment at specific genomic loci

  • Express as percentage of input or fold-enrichment over IgG control

  • Normalize to appropriate control regions

Each method offers different advantages depending on experimental goals, with western blot providing bulk measurement and microscopy/flow cytometry offering single-cell resolution.

What is the relationship between pH3T6 and other histone H3 phosphorylation sites in disease contexts?

Research in glioblastoma multiforme has revealed complex relationships between different H3 phosphorylation sites:

These findings highlight the complexity of histone modifications in disease and suggest multiple parallel pathways that could be targeted therapeutically.

What experimental designs best assess the functional consequences of pH3T6?

To comprehensively investigate pH3T6 function:

• Genetic approaches:

  • CRISPR/Cas9 to generate H3 T6A mutants (non-phosphorylatable)

  • CRISPR/Cas9 to generate H3 T6E mutants (phosphomimetic)

  • Compare phenotypes and transcriptional profiles

• Pharmacological approaches:

  • PKC inhibitors (like Enzastaurin) to reduce pH3T6

  • PKC activators to increase pH3T6

  • Time-course experiments to determine acute vs. chronic effects

• Combined ChIP-seq approaches:

  • pH3T6 ChIP-seq to map genomic distribution

  • RNA-seq after modulating pH3T6 levels

  • Integration with other histone modification data

• Protein interaction studies:

  • Mass spectrometry to identify pH3T6-specific "readers"

  • Co-IP experiments to validate interactions

  • In vitro binding assays with modified peptides

• Single-cell approaches:

  • CUT&Tag for pH3T6 with single-cell readout

  • Correlate with transcriptional heterogeneity