CARD8 Antibody

What is CARD8 and what experimental approaches can detect its different isoforms?

CARD8 belongs to the caspase recruitment domain (CARD)-containing family of proteins involved in pathways leading to caspase activation or nuclear factor kappa-B (NFKB) signaling. It functions as a component of the inflammasome, a protein complex that activates proinflammatory caspases.

Methodological approach:
Western blot analysis using CARD8 antibodies typically reveals multiple isoforms:

• Full-length CARD8 (~62 kDa)

• FIIND-processed CARD8 (~29 kDa)

• Alternative isoform (~49 kDa)

CARD8 protein contains a disordered amino-terminus, a FIIND domain, and a CARD domain that resembles NLRP1. For optimal detection, antibodies targeting the C-terminus can identify both full-length and processed forms .

What are the recommended experimental applications for CARD8 antibodies?

CARD8 antibodies can be utilized in multiple experimental applications:

Methodological consideration: Antigen retrieval methods significantly impact IHC results. For optimal staining, researchers should consider TE buffer pH 9.0 or citrate buffer pH 6.0 .

How is CARD8 expression distributed across human tissues?

CARD8 displays a broad tissue distribution pattern but with notable variations in expression levels:

Methodological findings: Transcriptome analysis from 95 individuals across 27 different human organs and tissues revealed CARD8 is widely expressed, with a tissue expression profile similar to NLRP1 . This broad expression pattern contrasts with other inflammasome receptors, which show more restricted expression patterns. CARD8 expression is particularly upregulated in various cancer types .

How does the human-specific F59-F60 motif in CARD8 facilitate inflammasome activation in response to HIV-1 infection?

The F59-F60 motif in human CARD8 represents a crucial site for HIV-1 protease (HIV-1 PR) cleavage, which activates the CARD8 inflammasome.

Methodological findings and approach:
Human CARD8 contains a unique F59-F60 motif that evolved specifically in humans after divergence from chimpanzees. While F59 (P1 site) is conserved among hominoids, gibbons, and Old World monkeys, only humans have phenylalanine at position 60 (P1' site) .

Experimental validation through site-directed mutagenesis demonstrated:

• Replacing F60 with leucine (F60L, found in chimpanzees/bonobos/gorillas) or serine (F60S, found in gibbons/Old World monkeys) significantly reduced HIV-1 PR cleavage efficiency

• CARD8 knockout (KO) THP-1 cells complemented with WT human CARD8 showed IL-1β secretion and cell death in response to HIV-1 infection

• Complementation with F60L, F60S, or F60A mutants failed to restore responsiveness to HIV-1

This motif emerged in the human lineage after divergence from the common ancestor with chimpanzees and is present in Neanderthal CARD8, dating its emergence to within the last million years .

What methodologies are optimal for investigating CARD8 knockout and its effects on inflammasome function?

Methodological approach for CARD8 KO generation and validation:

• CRISPR/Cas9-mediated knockout in cell lines (e.g., THP-1 cells)

• Validation through immunoblotting using antibodies specific to the CARD8 C-terminus

• Functional validation using known CARD8 agonists:

  • Val-boro-Pro (VbP) activates CARD8 inflammasome

  • Nigericin (which activates NLRP3 inflammasome) serves as a control to confirm inflammasome pathway specificity

Research findings from CARD8 KO studies:
CARD8 KO THP-1 cells showed:

• Abolished response to VbP

• Preserved response to nigericin (NLRP3 activator)

• Significantly reduced IL-1β secretion, cell death, and CASP1 activation following HIV-1 infection

• Similar reduction levels in both CARD8 KO and CASP1 KO cells, suggesting CARD8 is the primary inflammasome sensor for HIV-1 in THP-1 cells

How do CARD8 polymorphisms associate with inflammatory and autoimmune diseases?

CARD8 polymorphisms have been implicated in various inflammatory and autoimmune conditions.

Methodological approaches for investigating polymorphisms:

• DNA extraction from peripheral venous whole blood samples

• SNP selection based on minor allele frequency >0.05 in the target population

• Genotyping using MassARRAY or similar technologies

• PCR primer design using appropriate software (e.g., ADS2.0)

Research findings:
CARD8 polymorphism rs2043211 has been associated with:

• Decreased incidence of ileal Crohn's disease and stenotic/fistulizing CD

• Increased risk of gout in Chinese and European populations

• Decreased risk of ankylosing spondylitis in a Swedish study

Primer sequences for common CARD8 SNPs:

How does CARD8 regulate cytokine and chemokine expression in endothelial cells and atherosclerotic lesions?

CARD8 significantly influences inflammatory signaling in endothelial cells, potentially contributing to atherosclerosis pathophysiology.

Methodological findings:

• Endothelial and smooth muscle cells in arterial tissue express CARD8

• CARD8 expression correlates with vWF, CD163, and inflammatory genes (CXCL1, CXCL6, PDGF-A) in atherosclerotic plaques

• Experimental knockdown of CARD8 in human umbilical vein endothelial cells (HUVECs) significantly altered inflammatory proteins, including CXCL1, CXCL6, PDGF-A, MCP-1, and IL-6

Research implications: CARD8 appears to play a significant role in endothelial activation, suggesting potential therapeutic approaches targeting CARD8 in atherosclerosis and related inflammatory vascular conditions .

What is the temporal dynamics of CARD8 inflammasome activation following viral infection?

CARD8 inflammasome activation exhibits a distinct temporal pattern following HIV-1 infection.

Methodological approach and findings:
Time-course experiments revealed:

• CARD8-dependent increase in IL-1β as early as 2 hours post-infection

• Initial plateau for 6 hours followed by further increase at 24 hours post-infection

• Early activation (before reverse transcription and de novo viral protein synthesis) suggests CARD8 detects packaged HIV-1 PR released into target cells upon viral entry

• Treatment with HIV PR inhibitor lopinavir (LPV) blocked CARD8 inflammasome activation, confirming CARD8 senses HIV-1 PR activity

This biphasic activation pattern suggests CARD8 functions as an innate immune sensor at multiple stages of viral infection.

How does the interaction between CARD8 and other inflammasome components differ between humans and other species?

CARD8 exhibits significant species-specific differences in its inflammasome interactions.

Methodological findings:

• CARD8 lacks orthologues in rodents

• Human CARD8 T60 variant interacts with NLRP1 and negatively regulates NLRP1 inflammasome activation

• Chimpanzee CARD8 does not recognize proteases from HIV or simian immunodeficiency viruses (SIVcpz), while human CARD8 is cleaved by both HIV-1 PR and SIVcpz PR

• Specific CARD8 isoforms (T60 variant but not canonical T48 isoform) negatively regulate NLRP1 inflammasome activation by direct interaction with NLRP1

Research implication: These species-specific differences highlight the importance of caution when extrapolating CARD8 findings from animal models to humans.

What are the optimal storage and handling conditions for CARD8 antibodies?

For maximum stability and performance of CARD8 antibodies:

Methodological recommendations:

• Store at -20°C

• Stable for approximately one year after shipment

• Aliquoting is unnecessary for -20°C storage for small volumes

• PBS with 0.02% sodium azide and 50% glycerol pH 7.3 is the optimal storage buffer

• Avoid repeated freeze-thaw cycles

How can researchers validate CARD8 activation in experimental settings?

Methodological approaches to measure CARD8 activation:

• IL-1β secretion measurement: ELISA assays to detect mature IL-1β release

• Cell death assessment: LDH release, PI/Annexin V staining, or MTT assays

• Caspase-1 activation: FLICA assay or western blotting for cleaved caspase-1

• CARD8 cleavage detection: Western blot analysis with antibodies specific to the CARD8 C-terminus can identify the ~33 kDa cleavage product resulting from protease activity

• Proteasome-dependent activation: Proteasome inhibitors (e.g., bortezomib) can be used to confirm the proteasome dependence of CARD8 activation

Experimental controls:

• Use CARD8 KO cells as negative controls

• Include NLRP3 activators (e.g., nigericin) as inflammasome pathway controls

• Employ protease inhibitors (e.g., lopinavir for HIV-1 PR) to confirm protease-dependent activation