Phospho-FADD (Ser191) Antibody
Description
Introduction to Phospho-FADD (Ser191) Antibody
Phospho-FADD (Ser191) Antibody is a polyclonal rabbit antibody designed to specifically detect FADD (Fas-associated death domain protein) when phosphorylated at serine residue 191 (Ser191) in mouse models. This antibody is critical for studying FADD’s role in apoptosis signaling, particularly in death receptor pathways such as TNF receptor-mediated apoptosis . Its specificity ensures accurate detection of post-translational modifications (PTMs) that regulate FADD’s function in cellular death pathways.
Mechanism of Action
FADD acts as an adaptor protein in death receptor signaling, bridging receptors like Fas and TNF-R1 to caspase-8 at the death-inducing signaling complex (DISC) . Phosphorylation at Ser191 (or Ser194 in human FADD) modulates FADD’s subcellular localization and oligomerization state. In apoptosis, TNFα/ActD treatment induces FADD redistribution from the nucleus to the cytoplasm, with monomeric non-phosphorylated FADD accumulating in the cytosol to activate caspase-8 . The antibody enables visualization of these dynamic changes via techniques like immunoblotting (IB), immunohistochemistry (IHC), and immunofluorescence (IF).
Key Features of the Antibody
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Specificity: Detects only phosphorylated Ser191 FADD (mouse-specific; human counterpart targets Ser194) .
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Dilutions: WB (1:500–1:1000), IHC (1:50–1:100), ELISA (1:10,000) .
3.1. Role in Apoptosis Signaling
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TNFα/ActD-Induced Apoptosis: The antibody revealed that TNFα/ActD treatment reduces nuclear phosphorylated FADD (pFADD) while increasing cytoplasmic monomeric FADD. This redistribution depends on caspase-3 and -8 activation, suggesting a feed-forward mechanism to amplify apoptosis .
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T-Cell Lymphoblastic Lymphoma (T-LBL): In T-LBL, reduced FADD levels and altered Ser191 phosphorylation correlate with diminished apoptosis and tumor aggressiveness. IHC studies using this antibody identified two T-LBL subgroups based on pFADD levels, with the "Low" group showing nuclear pFADD depletion and cytoplasmic FADD loss .
3.2. Subcellular Localization Studies
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Nuclear vs. Cytoplasmic FADD: The antibody demonstrated that pFADD primarily resides in the nucleus under normal conditions, while cytoplasmic pFADD is enriched during apoptosis .
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Cross-Species Relevance: Human FADD phosphorylation at Ser194 (homologous to Ser191 in mouse) follows similar regulatory patterns, though species-specific antibodies are required for detection .
Research Implications
Product Specs
Target Background
- Overexpression of FADD and Caspase-8 suppresses proliferation while promoting apoptosis in human GBM cells. PMID: 28618251
- FADD expression and its phosphorylation serve as reliable biomarkers with prognostic value for T-cell lymphoblastic lymphoma stratification. PMID: 27556297
- FADD interference down-regulates Rheb expression and represses mTORC1 activity in breast cancer cell lines. FADD deficiency in MCF7 or MDA-231 cells induces autophagy, which can be rescued by recovering Rheb expression. PMID: 27013580
- Current data suggest FADD as a potential biomarker for pathological processes associated with the course of clinical dementia. PMID: 28320441
- At normal expression levels during bacterial infection, NleB1/NleB(CR) antagonizes death receptor-induced apoptosis of infected cells by modifying FADD in an irreversible manner. PMID: 28860194
- Using the tDED filament structure as a template, structural analyses reveal the interaction surfaces between FADD and caspase-8 and the distinct mechanisms of regulation by cFLIP and MC159 through comingling and capping, respectively. PMID: 27746017
- In myelodysplastic syndrome, FADD expression is regulated by SPAG6, which influences its interaction with TRAIL death receptors. PMID: 28393201
- High levels of FADD and caspase-8, but not caspase-3, were associated with an increased incidence of coronary events in subjects from the general population. PMID: 28302628
- Both Fas associated via death domain gene copy number amplification and high protein expression were significantly associated with lymph node metastasis and exhibited the shortest disease-free survival and overall survival. PMID: 27764170
- The autoinflammation-associated H443P nlrc4 mutant is altered in its interaction with SUG1 and ubiquitinated proteins, triggering constitutive caspase-8-mediated cell death dependent on FADD but independent of Ser(533) phosphorylation. PMID: 27974463
- This study demonstrates that C5a signaling induces apoptosis in brain vascular endothelial cells in experimental lupus through activation of FADD. PMID: 27213693
- Authors identify upregulated non-canonical nuclear factor-kappaB (NF-kappaB) signaling directly linked to the tumor necrosis with MT2A and pFADD genes. pFADD with MT2A can inhibit apoptosis and promote proliferation in colorectal cancer cells. PMID: 28061540
- Knockdown of cFLIPL and induced expression of FADD rapidly accumulate intracellular ROS accompanied by JNK1 activation, supporting apoptosis. PMID: 27619661
- Data indicate that FADD-mediated apoptotic cell death is directed by ubiquitination of cFLIPL and inhibition of NF-kappaB activation. PMID: 26972597
- Structural analysis of the roles of DR5 death domain mutations on oligomerization of the DR5 death domain-FADD complex in the death-inducing signaling complex formation has been presented. PMID: 26995783
- A20 targets caspase-8 and FADD to protect HTLV-I-infected cells. PMID: 26437781
- TCGA analysis showed that ANO1 and FADD, located at 11q13, were co-expressed at the transcript level and significantly associated with overall and disease-free survival. PMID: 26808319
- Data (including data obtained in transgenic mice) suggest FADD is key in the genesis of neural tube defects in pups of diabetic mice; unfolded protein response/endoplasmic reticulum stress was prevented by overexpression of human dominant negative FADD. PMID: 26419589
- The multifaceted kinase, CK2, phosphorylates FADD and is involved in its sub-cellular localization. PMID: 26253696
- The gene expression analysis showed statistically significant differences between cases and healthy controls for both FADD (p<0.02) and FAS (p<0.007) genes. PMID: 25129245
- Observed upregulation of cortical p-194 FADD and p-FADD/FADD ratio (higher pro-survival index) in major depression; this could play a major role in counteracting the known activation of the intrinsic (mitochondrial) apoptotic pathway in the brain. PMID: 25075716
- FADD death effector domain and c-FLIP death effector domain structures, the binding activity of FADD DED to the c-FLIP death effector domains, and the protein-protein interactions involved in the regulation of both apoptosis and necrosis have been described. PMID: 24355299
- The genotype of the promoter SNP (rs10898853) of FADD was found to be significantly associated with papillary thyroid cancer in a South Korean case-control study. PMID: 24434721
- These results indicate that FADD, as a host pro-apoptotic protein, plays important roles in regulating HIV-1 replication and production in several ways, and apoptotic pathway inhibition can decrease HIV-1 replication and production. PMID: 24752353
- Depletion of alphaNAC in multiple types of cancer cells induces typical apoptotic cell death. This anti-apoptotic function is mediated by the FADD/c-Jun N-terminal kinase pathway. PMID: 24901053
- High expression of FADD may be an independent biomarker for poor prognosis in nasopharyngeal carcinoma. PMID: 25305096
- Combined FADD, TMEM16A, and PPFIA1 gene expressions are associated with invasive ductal carcinoma of the breast. PMID: 24886289
- FADD elevation in leukocytes might be interpreted as the molecular equivalent of an elevated general inflammatory activity in relapsing remitting multiple sclerosis. PMID: 24603611
- Antagonizing miR-128a expression sensitized Jurkat/R cells to Fas-mediated apoptosis through derepression of FADD expression. PMID: 24316133
- Association between FADD protein expression in advanced-stage head and neck squamous cell carcinoma and clinicopathological features and outcome has been studied. PMID: 23763459
- Data show that calmodulin (CaM) binds to the death domain of Fas (FasDD) with an apparent dissociation constant (Kd) of ~2 muM and 2:1 CaM:FasDD stoichiometry. PMID: 23760276
- Data show that Pin1 prevents Fas-mediated apoptosis in activated eosinophils via interactions with phospho-FADD. PMID: 23606538
- Kashin-Beck disease patients have significantly increased FADD expression in the middle layer but decreased FLIP expression in the upper layer of the cartilage. PMID: 22126763
- Ubiquitination and degradation of the FADD adaptor protein regulate death receptor-mediated apoptosis and necroptosis. PMID: 22864571
- The FADD gene amplification was not useful for the predictive marker of cancerization but is possibly related to the malignancy of oral squamous cell carcinoma. PMID: 22838074
- This review discusses the possible link that could exist between the adenosine-dependent regulation of FADD in the inflammatory context of rheumatoid arthritis. PMID: 22253026
- FADD cleavage by NK cell granzyme M enhances its self-association to facilitate procaspase-8 recruitment for auto-processing leading to caspase cascade. PMID: 21979465
- Formation of hydrogen-bonded secondary structure in the C-terminal domain of the Fas-associated death domain has been investigated. PMID: 22130896
- DJ-1 protects against TRAIL-induced apoptosis through the regulation of death-inducing signaling complex (DISC) formation. PMID: 21785459
- Polo-like kinase 1 (Plk1) failed to phosphorylate the Aur-A-unphosphorylatable FADD substitution mutant S203A. PMID: 21978935
- An essential role of calmodulin in mediating Fas-induced FADD-independent activation of Src-ERK signaling pathways, which promote survival signaling in pancreatic cancer cells, has been identified. PMID: 21613217
- FADD: an endogenous inhibitor of RIP3-driven regulated necrosis. PMID: 21894190
- Our results suggest that deregulated miR-155 promotes Fas-mediated apoptosis in human intervertebral disc degeneration by targeting FADD and caspase-3. PMID: 21706480
- These data provide evidence that serine 194 phosphorylated Fas-associated death domain protein is involved in the proliferation of normal and neoplastic B cells and has features of a novel proliferation marker. PMID: 21315423
- FADD and TRIM21 together negatively regulate the late IFN-alpha pathway in response to viral infection. PMID: 21183682
- Our data demonstrate that in response to taxol, Plk1 endows death-promoting and tumor-suppressor functions to its substrate, FADD. PMID: 20890306
- Results suggest that the CD95-DD+CT:FADD-DD complex formed in solution is dissociated at lower pH. PMID: 20947025
- Findings show that the Fas-FADD death domain (DD) complex forms an asymmetric oligomeric structure composed of 5-7 Fas DD and 5 FADD DD, whose interfaces harbor associated autoimmune lymphoproliferative syndrome mutations. PMID: 20935634
- FADD is essential at early stages of hematopoiesis; its deletion with the Mx1-cre transgene in bone marrow cells leads to impairment of peripheral lymphoid, myeloid, and erythroid cell lineages. PMID: 21115735
- We describe here a complex clinical disorder, its genetic basis, and some of the key mechanisms underlying its pathogenesis. Our findings highlight the key role of FADD in Fas-dependent and Fas-independent signaling pathways in humans. PMID: 21109225
Q&A
What is FADD and why is the phosphorylated form significant in research?
FADD (Fas-Associated Death Domain protein) functions as an adaptor molecule that interacts with various cell surface receptors and mediates cell apoptotic signals. It plays a crucial role in the death signaling pathway initiated by receptors like TNFRSF6/Fas-receptor and tumor necrosis factor receptor. The phosphorylated form of FADD at Serine 191 represents a specific post-translational modification that has distinct biological functions compared to unphosphorylated FADD .
When studying cell death mechanisms, distinguishing between phosphorylated and non-phosphorylated FADD is essential as phosphorylation can regulate FADD's activity and interactions with other proteins in the apoptotic pathway. Phospho-specific antibodies enable researchers to detect these subtle but significant modifications that might play crucial roles in disease mechanisms and cellular processes.
What is the difference between Phospho-FADD (Ser191) and Phospho-FADD (Ser194)?
Phospho-FADD (Ser191) refers specifically to mouse FADD phosphorylated at serine position 191, while Phospho-FADD (Ser194) corresponds to human FADD phosphorylated at serine position 194 . This species-specific difference is critical when designing experiments:
| Species | Phosphorylation Site | Antibody Reactivity | Molecular Weight |
|---|---|---|---|
| Mouse | Serine 191 | Mouse-specific | 23-25 kDa |
| Human | Serine 194 | Human-specific | 28 kDa |
What are the optimal applications for Phospho-FADD (Ser191) Antibody?
Phospho-FADD (Ser191) Antibody has been validated for several applications with specific recommended dilutions:
| Application | Recommended Dilution | Notes |
|---|---|---|
| Western Blot (WB) | 1:500-1:2000 | Best for quantitative analysis of protein expression levels |
| Immunohistochemistry (IHC) | 1:50-1:300 | Suitable for tissue localization studies |
| ELISA | 1:5000-1:10000 | High sensitivity detection in solution |
For optimal results in Western blotting, researchers should use reducing conditions with appropriate buffer systems. When performing IHC, antigen retrieval methods may enhance detection sensitivity . Experimental validation of optimal antibody concentration is recommended for each specific application and sample type.
How can I optimize Western blot protocols for detecting phosphorylated FADD?
For successful Western blot detection of phosphorylated FADD, consider these methodological refinements:
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Sample Preparation: Include phosphatase inhibitors in lysis buffers to prevent dephosphorylation during sample processing.
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Loading Control: Use a total FADD antibody on parallel blots to normalize phospho-FADD signals.
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Membrane Selection: PVDF membranes often provide better results than nitrocellulose for phospho-protein detection.
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Blocking Solution: Use 5% BSA instead of milk, as milk contains casein phosphoproteins that may interfere with phospho-specific antibody binding.
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Antibody Dilution: Begin with a 1:500 dilution in BSA-based buffer and optimize as needed .
How can I validate the specificity of Phospho-FADD (Ser191) Antibody in my experimental system?
Validating antibody specificity is crucial for reliable research outcomes. For Phospho-FADD (Ser191) Antibody, consider implementing these validation strategies:
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Phosphatase Treatment: Treat duplicate samples with lambda phosphatase before Western blotting. The phospho-specific signal should disappear in treated samples.
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Immunizing Peptide Competition: Pre-incubate antibody with excess phosphopeptide immunogen to block specific binding sites.
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FADD Knockdown/Knockout Controls: Use FADD siRNA or CRISPR-edited cell lines lacking FADD expression as negative controls.
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Inducing Phosphorylation: Compare samples from conditions known to induce FADD phosphorylation (e.g., nocodazole or hydroxyurea treatment) with untreated controls .
These validation experiments should be accompanied by appropriate controls, including secondary antibody-only controls to rule out non-specific binding.
What cellular conditions modulate FADD phosphorylation at Ser191?
FADD phosphorylation status changes under various cellular conditions:
| Condition | Effect on FADD Phosphorylation | Experimental Model |
|---|---|---|
| Cell Cycle Arrest | Increased phosphorylation | Nocodazole (1 μg/mL) treatment for 20 hours |
| DNA Damage | Altered phosphorylation patterns | Hydroxyurea (4 mM) treatment for 20 hours |
| Apoptosis Induction | Dynamic phosphorylation changes | FAS ligand stimulation |
When designing experiments to study FADD phosphorylation dynamics, time-course studies are recommended to capture transient phosphorylation events. Additionally, co-immunoprecipitation experiments can reveal how phosphorylation affects FADD's interaction with binding partners in signaling complexes .
Why am I detecting non-specific bands in my Western blot using Phospho-FADD (Ser191) Antibody?
Non-specific bands are a common challenge when working with phospho-specific antibodies. Consider these methodological solutions:
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Antibody Titration: Test multiple antibody dilutions (1:500, 1:1000, 1:2000) to find the optimal signal-to-noise ratio.
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Blocking Optimization: Extend blocking time (2-3 hours at room temperature) or use alternative blocking agents like fish gelatin.
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Cross-Reactivity Analysis: Some non-specific bands may represent cross-reactivity with other phosphorylated proteins. Perform peptide competition assays with both target and non-target phosphopeptides.
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Sample Preparation: Ensure complete protein denaturation and consider additional purification steps for complex samples .
For polyclonal antibodies like the Phospho-FADD (Ser191) Antibody, some batch-to-batch variation is expected. When possible, validate critical results with alternative detection methods or antibodies from different suppliers.
How should I store and handle Phospho-FADD (Ser191) Antibody to maintain reactivity?
Proper storage and handling are essential for maintaining antibody performance:
| Storage Condition | Recommendation | Duration |
|---|---|---|
| Long-term | -20°C in aliquots | Up to 1 year |
| Working solution | 2-8°C | Up to 1 month |
| Shipping | 4°C with ice packs | - |
| Avoid | Repeated freeze-thaw cycles | No more than 3 cycles |
Most Phospho-FADD (Ser191) antibodies are supplied in a buffer containing 50% glycerol, 150mM NaCl, PBS (pH 7.4), and 0.02% sodium azide. When diluting, use fresh buffer with similar composition but without azide for applications sensitive to this preservative .
How can I design experiments to study the functional significance of FADD phosphorylation?
To investigate the biological role of FADD phosphorylation at Ser191, consider these experimental approaches:
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Site-Directed Mutagenesis: Generate phospho-mimetic (S191D/E) and phospho-null (S191A) FADD mutants for expression in FADD-knockout cells.
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Phosphorylation Dynamics: Use time-course experiments with apoptosis inducers to track changes in FADD phosphorylation status.
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Protein-Protein Interaction Studies: Perform co-immunoprecipitation experiments with wild-type FADD and phospho-mutants to identify phosphorylation-dependent interaction partners.
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Cellular Localization Studies: Use immunofluorescence to determine if phosphorylation affects FADD subcellular localization.
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Functional Assays: Compare apoptotic responses in cells expressing wild-type versus mutant FADD proteins.
These approaches can reveal whether FADD phosphorylation enhances or inhibits apoptotic signaling in specific cellular contexts.
What are the best cell models for studying FADD phosphorylation?
Selection of appropriate cell models is critical for studying FADD phosphorylation:
| Cell Line | Origin | Advantages | Considerations |
|---|---|---|---|
| Jurkat | Human T lymphocyte | Well-characterized FADD-dependent apoptosis | Human FADD (Ser194) |
| HeLa | Human cervical epithelial | Good for imaging and transfection | Shown to exhibit phospho-FADD localization to nuclei |
| HT-29 | Human colon adenocarcinoma | Documented phospho-FADD expression | May have altered apoptotic pathways |
| Primary mouse T cells | Mouse | Physiologically relevant | Requires Ser191-specific antibodies |
When using these models, it's important to consider baseline phosphorylation levels and the expression of proteins that may modulate FADD phosphorylation, such as casein kinase 1α (CK1α), which has been implicated in phosphorylating FADD .
How does FADD phosphorylation status correlate with disease progression or treatment response?
FADD phosphorylation may serve as a biomarker in various pathological conditions:
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Cancer: Altered FADD phosphorylation has been observed in multiple cancer types, potentially correlating with chemotherapy resistance.
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Inflammatory Diseases: Changes in FADD phosphorylation status may reflect altered cell death mechanisms in chronic inflammation.
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Neurodegenerative Disorders: Dysregulated apoptotic signaling, including FADD phosphorylation, has been implicated in neuronal cell death.
To study these correlations, researchers should design tissue microarray studies using Phospho-FADD antibodies with appropriate controls and quantification methods. Multiplexed immunofluorescence techniques can reveal co-localization with other disease markers or signaling proteins.
What are the emerging techniques for studying FADD phosphorylation beyond traditional antibody-based methods?
Advanced technologies are expanding our ability to study protein phosphorylation:
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Phosphoproteomics: Mass spectrometry-based approaches can quantify multiple phosphorylation sites simultaneously without antibody limitations.
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Proximity Ligation Assay (PLA): This technique allows visualization of protein interactions dependent on FADD phosphorylation status in situ.
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CRISPR-Cas9 Genome Editing: Generation of endogenously tagged FADD or phospho-site mutants provides physiologically relevant models.
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Biosensors: FRET-based reporters can monitor FADD phosphorylation dynamics in living cells in real-time.
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Single-Cell Analysis: Recent advances allow measurement of phosphorylation events at the single-cell level, revealing population heterogeneity.
These approaches complement traditional antibody-based detection methods and may provide deeper insights into the temporal and spatial regulation of FADD phosphorylation.
