Phospho-CASP8 (S347) Antibody

What is the significance of Caspase-8 phosphorylation at Serine 347 in cellular signaling pathways?

Caspase-8 phosphorylation at Serine 347 represents a critical post-translational modification that regulates this protein's function in apoptotic and non-apoptotic pathways. This specific phosphorylation site is located within the region spanning amino acids 313-362 of human Caspase-8 . Unlike tyrosine phosphorylation sites (such as Y380) that have been more extensively characterized, S347 phosphorylation provides an additional regulatory mechanism that affects caspase-8's catalytic activity and protein interactions.

Methodologically, researchers should approach S347 phosphorylation studies by examining:

• Changes in phosphorylation status during different cellular conditions (apoptosis induction, cell migration)

• Correlation with other post-translational modifications

• Effects on caspase-8 recruitment to the death-inducing signaling complex (DISC)

• Impact on downstream substrate cleavage patterns

The phosphorylation at S347 should be evaluated in context with other known regulatory phosphorylation sites (Y380, T273, S287, S305) to develop a comprehensive understanding of caspase-8 regulation .

What are the optimal experimental conditions for detecting Phospho-CASP8 (S347) in different sample types?

Detecting Phospho-CASP8 (S347) requires careful optimization of experimental conditions based on sample type and analysis method:

For Western Blotting:

• Recommended dilution range: 1:500-1:2000

• Sample preparation: Use phosphatase inhibitors in lysis buffers to prevent dephosphorylation

• Expected molecular weight: 55kDa for the full-length phosphorylated protein

• Blocking conditions: Use 5% BSA rather than milk (phospho-epitopes can be masked by casein)

For Immunohistochemistry:

• Recommended dilution range: 1:100-1:300

• Antigen retrieval: Citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

• Detection systems: Both chromogenic and fluorescent secondary antibodies are suitable

For ELISA:

• Recommended dilution: 1:5000

Researchers should note that these antibodies have been validated primarily for human and rat samples , and additional validation is required for other species.

How can researchers validate the specificity of a Phospho-CASP8 (S347) antibody?

Validating the specificity of Phospho-CASP8 (S347) antibodies requires multiple complementary approaches:

Essential validation experiments:

• Phosphatase treatment control:

  • Split cell lysate into two parts

  • Treat one with lambda phosphatase

  • Western blot should show signal reduction/elimination in treated samples

• Phospho-mimetic and phospho-deficient mutants:

  • Generate S347A (cannot be phosphorylated) and S347E/S347D (mimics phosphorylation)

  • Compare antibody reactivity with wild-type and mutant proteins

  • S347A should show reduced/no signal

• Stimulation experiments:

  • Treat cells with phosphorylation-inducing stimuli (e.g., death receptor ligands)

  • Monitor temporal changes in S347 phosphorylation status

• Knockdown/knockout controls:

  • Use CASP8-null cell lines (like 4KO HeLa cells mentioned in )

  • Reintroduce wild-type or mutant CASP8

  • Verify antibody specificity by signal absence in knockout cells

• Peptide competition:

  • Pre-incubate antibody with phosphorylated peptide immunogen (spanning S347)

  • Signal should be blocked if antibody is specific

These validation steps ensure reliable detection of true Phospho-CASP8 (S347) signal versus non-specific binding.

How does phosphorylation at S347 mechanistically affect caspase-8 dimerization and activation?

Caspase-8 activation involves a complex process of recruitment, dimerization, and autocatalytic processing. The effect of S347 phosphorylation on this process can be analyzed through sophisticated biochemical and structural approaches:

Experimental approaches to investigate this mechanism:

Research indicates that the dimerization/dissociation balance of caspase-8 is a critical regulator of apoptotic responses . While Y380 phosphorylation has been shown to interfere with autoproteolytic cleavage without affecting recruitment to the DISC or DED chain assembly , S347 phosphorylation may have distinct effects on the dimerization equilibrium.

The unique position of S347 in the linker region between the large and small catalytic subunits suggests it could affect the stability of processed dimers, potentially altering the equilibrium binding constant (Kd) of approximately 3.3 μM for caspase-8 dimers .

What is the relationship between caspase-8 S347 phosphorylation and its non-apoptotic functions in cell migration and cancer progression?

Caspase-8 exhibits paradoxical roles in cancer, functioning as both a tumor suppressor through its apoptotic activity and a promoter of cancer progression through non-apoptotic functions. The relationship between S347 phosphorylation and these non-apoptotic functions is complex:

Methodological approach to investigate this relationship:

• Migration and invasion assays:

  • Compare cells expressing wild-type caspase-8 versus S347A and S347E mutants

  • Measure effects on focal adhesion dynamics, migration velocity, and invasiveness

  • Evaluate response to integrin ligation

• Protein-protein interaction analysis:

  • Perform immunoprecipitation of wild-type versus phospho-mutant caspase-8

  • Identify differential interaction partners, focusing on focal adhesion complex components

  • Research shows caspase-8 interacts with focal adhesion kinase (FAK) and calpain 2 (CPN2)

• In vivo metastasis models:

  • Generate stable cell lines expressing different caspase-8 phospho-variants

  • Assess metastatic potential in animal models

  • Evidence suggests caspase-8 can promote metastasis when apoptosis is compromised

Current research indicates that caspase-8 catalytic activity is not required for promoting cell migration; rather, caspase-8 serves as a scaffold protein interacting with the focal adhesion complex . Phosphorylation at S347 may regulate these scaffold functions distinctly from its effects on apoptotic activity.

How do different phosphorylation events on caspase-8 integrate to form a comprehensive regulatory code?

Caspase-8 function is regulated by multiple phosphorylation events at different sites, including Y273, Y293, Y380, Y448, S287, S305, and S347 . These modifications form a complex regulatory code that determines caspase-8's functional output:

Methodological approaches to decipher this phosphorylation code:

• Mass spectrometry-based phosphoproteomics:

  • Monitor global phosphorylation patterns of caspase-8 under different stimuli

  • Identify co-occurring or mutually exclusive phosphorylation events

  • Quantify stoichiometry of different phosphorylation sites

• Mutational analysis with combinatorial phospho-site mutations:

  • Generate caspase-8 variants with multiple phospho-sites mutated

  • Compare functional consequences to single-site mutations

  • Assess hierarchical relationships between different phosphorylation events

• Kinase and phosphatase identification:

  • Use kinase inhibitor screens and in vitro kinase assays to identify regulators

  • Current research indicates Polo-like kinase 3 (Plk3) phosphorylates T273 , and Src family kinases target Y380

  • Identify kinases specifically targeting S347

• Temporal dynamics analysis:

  • Track phosphorylation changes at multiple sites using site-specific antibodies

  • Establish temporal sequence of phosphorylation/dephosphorylation events

  • Correlate with functional outcomes (apoptosis, migration, other non-apoptotic functions)

A comprehensive understanding of this phosphorylation code is crucial for therapeutic interventions targeting caspase-8 in cancer and inflammatory diseases.

What methodologies are most effective for studying caspase-8 S347 phosphorylation in the context of its DISC recruitment and activation?

The Death-Inducing Signaling Complex (DISC) formation is a critical step in extrinsic apoptosis initiation. Studying S347 phosphorylation in this context requires specialized methodologies:

Effective methodological approaches:

• DISC isolation and analysis:

  • Immunoprecipitate DISC components after death receptor stimulation

  • Use phospho-specific antibodies to detect S347 phosphorylation status within the DISC

  • Compare with total caspase-8 levels in the DISC

  • Research indicates DISC formation leads to DED-mediated oligomerization of procaspase-8

• Super-resolution microscopy:

  • Use dual-color super-resolution techniques to visualize phospho-S347 caspase-8 localization

  • Track recruitment to membrane-associated DISC complexes

  • Monitor spatial organization and clustering

• In vitro reconstitution systems:

  • Reconstitute DISC components with recombinant proteins

  • Include wild-type and phosphomimetic S347E caspase-8

  • Measure impact on caspase-8 activation kinetics and substrate processing

• Proximity ligation assays (PLA):

  • Detect interactions between phospho-S347 caspase-8 and other DISC components

  • Quantify changes in interaction frequency under different conditions

Current research shows that caspase-8 forms unidirectional filaments upon DISC assembly, with approximately six procaspase-8 molecules binding to a single FADD protein . The effect of S347 phosphorylation on this oligomerization process and subsequent activation steps remains to be fully characterized.

How can researchers distinguish between the effects of caspase-8 S347 phosphorylation on apoptosis versus necroptosis regulation?

Caspase-8 plays dual roles in cell death regulation: initiating apoptosis and inhibiting necroptosis. Distinguishing the effects of S347 phosphorylation on these pathways requires careful experimental design:

Methodological approach:

• Cell death modality discrimination:

  • Use live-cell imaging with multiple death markers:

    • Annexin V (apoptosis)

    • Propidium iodide uptake (membrane permeability)

    • MLKL phosphorylation (necroptosis)

  • Compare wild-type, S347A, and S347E caspase-8 expressing cells

• Genetic manipulation of the necroptotic machinery:

  • Combine caspase-8 phospho-variants with RIPK1/RIPK3/MLKL knockdown or inhibition

  • Recent research shows caspase-8 prevents tissue damage by inhibiting necroptosis

  • Test whether S347 phosphorylation affects caspase-8's ability to cleave RIPK1 at Asp-324

• Analysis of RIP kinase complexes (necrosomes):

  • Immunoprecipitate RIPK1/RIPK3 complexes

  • Assess incorporation of different caspase-8 phospho-variants

  • Evaluate impact on necrosome formation and activity

• In vivo models:

  • Generate knock-in mouse models with S347A phospho-deficient mutation

  • This approach revealed that preventing T265 phosphorylation of caspase-8 protected against TNF-induced necroptotic cecum damage but increased injury in the small intestine

  • Similar studies with S347 mutations could reveal tissue-specific roles

The interplay between phosphorylation at S347 and caspase-8's ability to form heterodimers with FLIP(L), which is important for necroptosis inhibition , represents a particularly important area for investigation.

What are the most common technical issues when using Phospho-CASP8 (S347) antibodies and how can they be resolved?

Researchers commonly encounter several technical challenges when working with Phospho-CASP8 (S347) antibodies:

Storage recommendations: Store at -20°C for up to one year . The antibody formulation typically includes 50% glycerol, 0.5% BSA, and 0.02% sodium azide to maintain stability .

How can researchers optimize protocols for detecting low levels of Phospho-CASP8 (S347) in clinical samples?

Detecting low-abundance phospho-proteins like Phospho-CASP8 (S347) in clinical samples presents unique challenges requiring optimized protocols:

Methodological optimization strategies:

• Sample preservation:

  • Process samples immediately after collection

  • Use phosphatase inhibitor cocktails with both Ser/Thr and Tyr phosphatase inhibitors

  • Consider specialized preservation methods (PAXgene for tissue, phospho-specific fixatives)

• Signal amplification techniques:

  • Tyramide signal amplification (TSA) for immunohistochemistry

  • Proximity ligation assay (PLA) for increased sensitivity in tissue sections

  • Capillary-based immunoassays (e.g., WES Simple Western) for quantitative detection with minimal sample input

• Enrichment strategies:

  • Phospho-protein enrichment using metal oxide affinity chromatography (MOAC)

  • Immunoprecipitation with total caspase-8 antibody followed by phospho-specific detection

  • Two-step enrichment: caspase-8 IP followed by phospho-peptide enrichment

• Detection optimization for Western blot:

  • Extended transfer times for larger proteins

  • PVDF membranes (higher protein binding capacity than nitrocellulose)

  • Enhanced chemiluminescence substrates with extended signal duration

  • Optimal antibody dilution determined empirically (starting with 1:500)

• Immunohistochemistry enhancement:

  • Heat-induced epitope retrieval optimization

  • Amplification systems (e.g., HRP-conjugated polymers)

  • Chromogenic signal development optimization (time, temperature)

  • Start with 1:100 dilution and titrate as needed

What controls and standards should be included when using Phospho-CASP8 (S347) antibodies for quantitative applications?

Reliable quantitative analysis of Phospho-CASP8 (S347) requires rigorous controls and standards:

Essential controls and standards:

• Positive controls:

  • Cells treated with stimuli known to induce S347 phosphorylation

  • Recombinant phosphorylated caspase-8 protein (commercially available or lab-generated)

  • Phosphomimetic S347E/D caspase-8 expression

• Negative controls:

  • Phosphatase-treated samples

  • S347A mutant caspase-8 expression

  • Caspase-8 knockout/knockdown cells (such as 4KO HeLa cells)

• Antibody controls:

  • Primary antibody omission

  • Isotype control antibody (rabbit IgG)

  • Peptide competition with phosphorylated and non-phosphorylated peptides

• Normalization standards:

  • Total caspase-8 detection in parallel samples

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

  • Housekeeping proteins for immunohistochemistry

• Quantification standards:

  • Standard curves using recombinant phosphorylated protein

  • Inclusion of common sample across all experiments as inter-assay calibrator

  • Digital image analysis with defined threshold settings

• Sample processing controls:

  • Time-course of sample processing to monitor phosphorylation stability

  • Split samples processed with different protocols to assess method effects

  • Freeze-thaw cycle testing to determine stability limits

For Western blot quantification, researchers should note that the observed band for phosphorylated caspase-8 is typically at 55kDa , though a 38kDa band may also be present depending on processing status .

How does Phospho-CASP8 (S347) status correlate with cancer progression and therapeutic resistance?

Caspase-8 plays complex roles in cancer with both tumor-suppressive and tumor-promoting functions. The phosphorylation status at S347 may provide important insights into these dual roles:

Research approaches to investigate this correlation:

• Tissue microarray analysis:

  • Compare Phospho-CASP8 (S347) levels across tumor stages and grades

  • Correlate with patient outcomes and treatment responses

  • Research indicates that in certain cancers, high levels of caspase-8 expression may correlate with worse prognosis

• Cell line panels with varying therapeutic resistance:

  • Profile S347 phosphorylation status across sensitive and resistant lines

  • Assess changes in phosphorylation following treatment

  • Determine whether modulation of S347 phosphorylation alters therapeutic sensitivity

• Functional studies with phospho-variants:

  • Express S347A or S347E in cancer cell lines

  • Assess impact on:

    • Apoptotic sensitivity

    • Migration and invasion (caspase-8 has been shown to promote metastasis when apoptosis is compromised)

    • Response to standard therapeutics

• Mechanisms of therapeutic resistance:

  • Evaluate whether S347 phosphorylation affects:

    • Caspase-8's interaction with FLIP proteins (known to confer therapeutic resistance)

    • Caspase-8's non-apoptotic functions in promoting NF-κB activation and cytokine production

    • DNA repair mechanisms (caspase-8 has been implicated in promoting DNA repair)

Research has shown that Src-mediated Y380 phosphorylation leads to increased resistance to CD95-induced apoptosis . Similar investigations into S347 phosphorylation could reveal additional mechanisms of apoptosis resistance in cancer cells.

What methodological approaches can evaluate the role of caspase-8 S347 phosphorylation in inflammatory and neurodegenerative disorders?

Caspase-8 functions extend beyond cancer to inflammatory and neurodegenerative conditions. Methodological approaches to study S347 phosphorylation in these contexts include:

Research methodologies:

• Neuroinflammation models:

  • Analyze S347 phosphorylation in microglia activation states

  • Compare phosphorylation patterns in neurodegenerative disease models

  • Research indicates caspase-8 was detected in the insoluble fraction of affected brain regions from Huntington disease patients

• Patient-derived samples:

  • Analyze S347 phosphorylation in:

    • CSF from neurodegenerative disease patients

    • Post-mortem brain tissue with phospho-specific IHC

    • Peripheral blood mononuclear cells during inflammatory conditions

  • Compare with age-matched controls

• Single-cell analysis techniques:

  • Single-cell phospho-protein analysis in heterogeneous tissue samples

  • Spatial transcriptomics combined with phospho-protein detection

  • Cell-type specific responses to inflammatory stimuli

• Kinase inhibitor studies:

  • Test effects of specific kinase inhibitors on S347 phosphorylation

  • Assess functional consequences in inflammation models

  • Evaluate potential therapeutic approaches targeting this phosphorylation

• In vivo models with phospho-mutants:

  • Generate knock-in mouse models with S347A mutations

  • Assess impact on:

    • Neuroinflammatory responses

    • Neurodegeneration progression

    • Inflammatory disease susceptibility

  • Recent research with T265A mutation showed tissue-specific effects on TNF-induced damage

Understanding the role of S347 phosphorylation in these contexts could reveal new therapeutic targets for both inflammatory and neurodegenerative conditions.

How can researchers integrate Phospho-CASP8 (S347) analysis into multi-parameter studies of cell death pathways?

Modern cell death research recognizes the complex interplay between different death modalities (apoptosis, necroptosis, pyroptosis). Integrating Phospho-CASP8 (S347) analysis into multi-parameter studies requires sophisticated approaches:

Methodological integration strategies:

• Multiplexed immunofluorescence panels:

  • Combine Phospho-CASP8 (S347) with markers for:

    • Apoptosis (cleaved caspase-3, PARP)

    • Necroptosis (phospho-MLKL, RIPK1/3)

    • Pyroptosis (GSDMD, ASC specks)

    • FLIP expression (modulates caspase-8 function)

  • Use spectral unmixing for analyzing multiple fluorophores

• Flow cytometry and mass cytometry (CyTOF):

  • Develop panels including Phospho-CASP8 (S347) with death markers

  • Perform single-cell analysis of heterogeneous populations

  • Identify cell subsets with distinct phosphorylation patterns

• Live-cell imaging approaches:

  • Generate fluorescent reporters for caspase-8 activity

  • Combine with S347 phosphorylation-sensitive biosensors

  • Monitor temporal dynamics of phosphorylation and activation

• Integrated omics approaches:

  • Combine phosphoproteomics with:

    • Transcriptomics (mRNA expression patterns)

    • Metabolomics (metabolic changes during cell death)

    • Interactomics (protein interaction networks)

  • Apply systems biology modeling to interpret complex datasets

• In situ detection methods:

  • Multiplex immunohistochemistry for tissue samples

  • RNAscope combined with phospho-protein detection

  • Spatial proteomics to map phosphorylation events in tissue context