CRADD Antibody

What is CRADD and what is its molecular and functional profile?

CRADD (CASP2 and RIPK1 Domain Containing Adaptor with Death Domain), also known as RAIDD, is an apoptotic adaptor molecule specific for caspase-2 and FASL/TNF receptor-interacting protein RIP . The protein contains a death domain (DD) motif and plays crucial roles in cell death pathways and inflammation regulation.

CRADD has a calculated molecular weight of approximately 23 kDa, though it is typically observed at 22-23 kDa on Western blots . The protein is localized in both the cytoplasm and nucleus , suggesting diverse cellular functions beyond its well-known role in apoptosis.

The full amino acid sequence of human CRADD consists of 199 amino acids:
MEARDKQVLRSLRLELGAEVLVEGLVLQYLYQEGILTENHJQEINAQTTGLRKTMLLLDILPSRGPKAFDTFLDSLQEFPWVREKLKKAREEAMTDLPAGDRLTGIPSHILNSSPSDRQINQLAQRLGPEWEPMVLSLGLSQTDIYRCKANH PHNVQSQVVEAFIRWRQRFGKQATFQSLHNGLRAVEVDPSLLLHMLE

This protein is expressed in various human tissues and cell types, including endothelial cells, where it has been documented to function as an inducible suppressor of BCL10, a key mediator of responses to proinflammatory agonists .

What applications can CRADD antibodies be used for in laboratory research?

CRADD antibodies have been validated for multiple research applications as summarized in the following table:

For each application, researchers should perform antibody titration in their specific experimental system to determine optimal conditions. Positive controls include K-562 cells, 293T cells, and HepG2 cells for Western blot applications .

How does CRADD function in endothelial cells and inflammatory responses?

CRADD plays a critical regulatory role in endothelial cell homeostasis and inflammatory responses. Studies have revealed that CRADD functions as an inducible suppressor of BCL10, which is a key mediator of responses to proinflammatory agonists in endothelial cells .

In endothelial cells, CRADD attenuates responses to proinflammatory agonists. Research has demonstrated that CRADD-deficient cells display more F-actin polymerization with concomitant disruption of adherens junctions . This suggests that CRADD is essential for maintaining proper barrier function in endothelial cells.

Experimental approaches have shown that:

• Reduction of CRADD expression in murine endothelial cells with shRNA leads to increased sensitivity to inflammatory signals

• Analysis of microvascular endothelial cells isolated from CRADD-deficient mice shows compromised barrier function

• Intracellular delivery of recombinant cell-penetrating CRADD protein (CP-CRADD) can restore endothelial barrier function and suppress the induction of IL-6 and MCP-1 evoked by LPS and thrombin

These findings indicate that CRADD serves as a protective factor in maintaining the permeability barrier of endothelial cells, particularly lung microvascular endothelial cells (LMEC).

What are the optimal protocols for detecting CRADD-protein interactions?

For studying CRADD-protein interactions, a combination of co-immunoprecipitation and immunoblotting techniques has proven effective. Based on published methodologies, the following protocol is recommended:

• Cell lysis preparation:

  • Harvest cells and lyse in RIPA buffer supplemented with protease inhibitors

  • Clear lysates by centrifugation (typically 14,000 × g for 15 minutes at 4°C)

• Co-immunoprecipitation:

  • Incubate cleared lysates with specific antibodies against the interaction partner of interest (e.g., IRAK-1, BCL10)

  • Add protein A/G-agarose beads (e.g., from Thermo) and incubate with rotation at 4°C overnight

  • Wash immunoprecipitates thoroughly (typically 3-4 times with lysis buffer)

• Immunoblot analysis:

  • Resolve immunoprecipitates by SDS-PAGE and transfer to appropriate membranes

  • Probe with antibodies to CRADD (recommended dilution 1:500-1:2000)

  • Include appropriate controls, such as GAPDH, β-actin, or TATA-binding protein (TBP) for normalization

  • Analyze results using quantitative infrared imaging systems (e.g., LI-COR Odyssey)

For detecting the interaction between CRADD and BCL10 specifically, researchers have successfully immunoprecipitated complexes using antibodies to IRAK-1 followed by immunoblotting for BCL10 and CRADD .

How can researchers optimize CRADD antibody usage for subcellular localization studies?

Since CRADD localizes to both cytoplasmic and nuclear compartments , proper experimental design is critical for accurate subcellular localization studies:

• Subcellular fractionation approach:

  • Perform careful subcellular fractionation to separate cytoplasmic and nuclear fractions

  • Verify fraction purity using compartment-specific markers (e.g., GAPDH or β-actin for cytoplasm; TATA-binding protein (TBP) for nucleus)

  • Use Western blotting with CRADD antibodies at 1:500-1:2000 dilution on separate fractions

• Immunofluorescence microscopy approach:

  • Fix cells with paraformaldehyde (typically 4%)

  • Permeabilize with appropriate detergent (0.1% Triton X-100 for total cell permeabilization; digitonin for selective plasma membrane permeabilization)

  • Block with BSA or serum-containing buffer

  • Incubate with CRADD antibodies at optimized dilutions

  • Use fluorescently-labeled secondary antibodies

  • Co-stain with appropriate subcellular markers (e.g., DAPI for nucleus)

  • Image using confocal microscopy for precise localization

• Validation strategies:

  • Compare results using multiple antibodies targeting different epitopes of CRADD

  • Include CRADD-deficient cells or knockdown controls

  • Validate results using both fractionation and imaging approaches

These approaches allow for comprehensive analysis of CRADD localization patterns under different cellular conditions and in response to various stimuli.

What strategies should be employed to study CRADD's role in regulating BCL10-dependent pathways?

Based on research showing CRADD's function as a suppressor of BCL10-mediated inflammatory signaling , the following methodological approaches are recommended:

• Genetic manipulation approaches:

  • Generate CRADD-deficient cells using shRNA knockdown or CRISPR-Cas9 technology

  • Create CRADD-overexpressing cell lines for gain-of-function studies

  • Utilize cells from CRADD-deficient mice for primary cell analysis

• Recombinant protein delivery system:

  • Employ cell-penetrating CRADD protein (CP-CRADD) treatment

  • Compare with non-cell-penetrating CRADD as a control

  • Verify intracellular delivery by proteinase K treatment to remove surface-bound protein, followed by immunoblot analysis

• Functional readouts:

  • Measure endothelial barrier function using permeability assays

  • Assess F-actin polymerization and adherens junction integrity

  • Quantify proinflammatory cytokine production (e.g., IL-6, MCP-1)

  • Analyze NFκB pathway activation through nuclear translocation assays

• Protein-protein interaction studies:

  • Examine CRADD-BCL10 interactions under various inflammatory stimuli

  • Investigate CARMA3 signalosome assembly dynamics

  • Study IRAK-1 complex formation in the presence/absence of CRADD

These comprehensive approaches allow for detailed mechanistic studies of how CRADD regulates BCL10-dependent inflammatory pathways in endothelial and other cell types.

What controls are essential when using CRADD antibodies in experimental workflows?

Proper controls are critical for ensuring reliable results when working with CRADD antibodies:

• Positive controls for Western blotting:

  • Verified CRADD-expressing cell lines such as K-562, HEK-293, HeLa, MCF-7, or HepG2

  • Recombinant CRADD protein at known concentrations for standard curves

  • Tissue samples with confirmed CRADD expression (mouse heart, kidney, liver, skeletal muscle, testis)

• Negative controls:

  • CRADD knockout or knockdown cells/tissues

  • Isotype control antibodies (matched to the CRADD antibody's host species and isotype)

  • Secondary antibody-only controls to assess non-specific binding

• Specificity controls:

  • Peptide competition assays using the immunizing peptide/protein

  • Multiple antibodies targeting different CRADD epitopes

  • Cross-validation with orthogonal methods (e.g., mass spectrometry)

• Normalization controls:

  • Loading controls such as GAPDH, β-actin for whole cell or cytoplasmic fractions

  • TATA-binding protein (TBP) for nuclear fractions

  • Total protein normalization methods (e.g., stain-free gels, Ponceau S)

• Application-specific controls:

  • For immunoprecipitation: IgG control pulldowns

  • For IHC/IF: Isotype-matched control antibodies on serial sections

  • For endothelial cell studies: Both CRADD+/+ and CRADD-/- primary endothelial cells

Implementing these controls ensures experimental rigor and facilitates accurate interpretation of results when studying CRADD expression and function.

How should researchers design experiments to study CRADD's role in endothelial barrier function?

Based on published methodologies , a comprehensive experimental design for studying CRADD's role in endothelial barrier function should include:

• Cell models:

  • Primary lung microvascular endothelial cells (LMEC) from both CRADD+/+ and CRADD-/- mice

  • Human endothelial cell lines with CRADD knockdown/knockout

  • Endothelial cells treated with recombinant cell-penetrating CRADD (CP-CRADD)

• Barrier function assessments:

  • Transendothelial electrical resistance (TEER) measurements

  • Permeability assays using labeled dextrans or albumin

  • Real-time monitoring of barrier integrity using impedance-based systems

• Inflammatory stimulation:

  • Challenge cells with lipopolysaccharide (LPS)

  • Thrombin treatment to induce barrier disruption

  • Cytokine stimulation (TNF-α, IL-1β)

• Molecular analyses:

  • F-actin polymerization assessment via fluorescent phalloidin staining

  • Adherens junction integrity via VE-cadherin immunostaining

  • BCL10 expression and localization analysis

  • NFκB pathway activation measurement

• Functional rescue experiments:

  • Dose-response studies with CP-CRADD to restore barrier function

  • Timing experiments to determine optimal intervention points

  • Combined interventions targeting multiple pathway components

This comprehensive approach allows researchers to establish causality between CRADD expression levels and endothelial barrier function while elucidating the underlying molecular mechanisms.

How can researchers troubleshoot inconsistent detection of CRADD in different experimental systems?

Several factors can contribute to inconsistent CRADD detection. A systematic troubleshooting approach includes:

• Antibody-related issues:

  • Verify antibody specificity using positive controls (K-562, HEK-293, HeLa cells)

  • Test multiple antibodies targeting different epitopes

  • Optimize antibody concentration (recommended 1:500-1:2000 for Western blot)

  • Ensure proper storage and handling of antibodies

• Sample preparation concerns:

  • Ensure complete cell lysis with appropriate buffers containing protease inhibitors

  • For nuclear detection, use specialized nuclear extraction protocols

  • Consider the impact of post-translational modifications on epitope accessibility

  • Check protein degradation by including freshly prepared samples

• Technical variables:

  • Optimize protein loading amounts (typically 20-50 μg for Western blot)

  • Adjust transfer conditions for efficient protein transfer

  • Consider native versus denaturing conditions based on epitope characteristics

  • Test different blocking agents to reduce background

• Application-specific considerations:

  • For IHC: Test different antigen retrieval methods (TE buffer pH 9.0 or citrate buffer pH 6.0)

  • For IP: Increase antibody amount (0.5-4.0 μg) or lysate concentration (1.0-3.0 mg)

  • For IF: Optimize fixation and permeabilization conditions

• Biological variables:

  • Consider cell type-specific expression levels

  • Account for stimulus-dependent changes in expression

  • Check for potential isoform expression differences

  • Evaluate subcellular localization patterns

By systematically addressing these factors, researchers can improve the consistency and reliability of CRADD detection across different experimental systems.

What are the key considerations when interpreting CRADD expression data in relation to inflammatory pathways?

When interpreting CRADD expression data in inflammatory contexts, researchers should consider several critical factors:

• Context-dependent functions:

  • CRADD functions as an inducible suppressor of BCL10 in endothelial cells

  • Its role may differ between cell types (e.g., immune cells versus endothelial cells)

  • Consider both pro-apoptotic and anti-inflammatory functions

• Expression dynamics:

  • Evaluate both baseline and stimulus-induced expression patterns

  • Consider temporal dynamics following inflammatory stimulation

  • Assess both protein and mRNA expression levels to identify regulatory mechanisms

• Pathway cross-talk:

  • CRADD interacts with multiple signaling molecules (IRAK-1, BCL10)

  • Consider the CARMA3 signalosome assembly and function

  • Evaluate NFκB pathway activation in relation to CRADD levels

• Causality versus correlation:

  • Use genetic models (knockdown/knockout) to establish causality

  • Complement with gain-of-function approaches (CP-CRADD treatment)

  • Consider compensatory mechanisms in chronic CRADD deficiency

• Translational relevance:

  • Connect molecular findings to functional outcomes (e.g., barrier function)

  • Consider the relevance to disease models or human pathology

  • Evaluate potential for therapeutic targeting

By carefully considering these factors, researchers can develop more nuanced interpretations of CRADD expression data and its significance in inflammatory pathways.

How can CRADD antibodies be employed in studies of autoimmune and inflammatory disorders?

CRADD's role in regulating inflammation makes it a relevant target for autoimmune research. The following methodological approaches are recommended:

• Expression profiling in disease states:

  • Compare CRADD expression levels in affected versus healthy tissues

  • Correlate expression with disease severity metrics

  • Assess subcellular localization changes in disease states

• Functional studies in autoimmune models:

  • Analyze barrier function in models of vascular inflammation

  • Evaluate inflammatory cytokine production in CRADD-deficient systems

  • Assess autoantibody production in relation to CRADD function

• Therapeutic potential assessment:

  • Test CP-CRADD as a potential barrier-protective intervention

  • Evaluate combinations with established anti-inflammatory agents

  • Develop targeted approaches to modulate CRADD-BCL10 interactions

• Biomarker development:

  • Assess CRADD as a potential biomarker for vascular inflammation

  • Develop multiplexed assays combining CRADD with other inflammatory markers

  • Evaluate CRADD in longitudinal studies of inflammatory disease progression

These approaches can provide valuable insights into CRADD's role in autoimmune pathology and identify potential therapeutic interventions.