C9orf89 Antibody

What is C9orf89 and what are its functional roles in cellular signaling?

C9orf89, also known as CARD19 or BinCARD, is a mitochondria-localized protein that functions as a regulatory component in immune signaling pathways. It contains a caspase recruitment structural domain (CARD) that enables it to inhibit BCL10-induced NF-κB activation. While it plays a documented role in T cell receptor (TCR) signaling, deletion of endogenous CARD19 has relatively modest effects on Bcl10-dependent NF-κB activation compared to overexpression studies. The protein is expressed across multiple cell types and participates in the regulation of inflammatory and immune responses .

Recent clarification of the splicing pattern and sequence of the mRNA product from the C9orf89 gene has revealed important distinctions between isoforms. BinCARD2, comprising 183 amino acids, is now recognized as the properly spliced and most abundant isoform, while BinCARD1 represents an incompletely derived form .

What are the key structural and molecular characteristics of C9orf89?

C9orf89 protein exhibits the following molecular characteristics:

The difference between calculated and observed molecular weight suggests post-translational modification or specific protein processing that researchers should consider when performing western blot analysis .

What are the recommended experimental conditions for using C9orf89 antibodies?

Based on multiple commercial antibodies and published research protocols, the following conditions are recommended for optimal results:

Western Blot (WB):

• Dilution range: 1:500 - 1:4000 (antibody-dependent)

• Positive detection in: A549 cells, C2C12 cells, mouse kidney tissue, rat kidney tissue

• Expected band size: 18-20 kDa

Immunohistochemistry (IHC):

• Dilution range: 1:50 - 1:100 (polyclonal antibodies)

ELISA:

• Dilution: Up to 1:40000 (highly sensitive antibodies)

It is strongly recommended to titrate the antibody in each specific testing system to obtain optimal results, as performance can be sample-dependent .

What storage conditions are optimal for maintaining C9orf89 antibody activity?

For maximum stability and activity retention, C9orf89 antibodies should be:

• Stored at -20°C

• Aliquoted to avoid repeated freeze/thaw cycles

• Diluted in recommended buffers (typically PBS with 0.02% sodium azide and 50% glycerol pH 7.3)

Most C9orf89 antibodies demonstrate stability for one year after shipment when stored properly. For 20μl size preparations, some formulations contain 0.1% BSA for additional stabilization .

How does C9orf89/CARD19 modulate immune signaling pathways and what are the implications for autoimmunity research?

C9orf89/CARD19 plays a sophisticated regulatory role in immune signaling by modulating the TAK1/NF-κB pathway. Research has revealed that CARD19 strongly interacts with TAK1, but not with other components of the CBM signalosome. This interaction enables CARD19 to inhibit TAB2-mediated TAK1 activation and subsequent NF-κB activation .

Critically, CARD19 deficiency prevented Bm12-induced experimental systemic lupus erythematosus, suggesting potential therapeutic implications. This demonstrates that CARD19 negatively regulates BCR/TAK1-induced NF-κB activation, and its deficiency enhances B-cell tolerance to self-antigens .

What methodological approaches are most effective for validating C9orf89 knockout models?

Effective validation of C9orf89 knockout models requires a multi-faceted approach:

• Genotyping PCR: The genotype should be determined using PCR with specific primers designed to detect the targeted modification in the C9orf89 gene .

• Expression validation: Assessment of Card19 expression in tissues from wild-type and CARD19-deficient mice should be performed using:

  • Quantitative real-time PCR (qPCR)

  • Immunoblot with a CARD19-specific polyclonal antibody

  • Normalization using appropriate reference genes (e.g., β2-microglobulin for qPCR)

• Functional validation: This can include:

  • NF-κB pathway activity assessment

  • Evaluation of downstream gene expression changes through RNA sequencing

  • Phenotypic characterization through immune challenge models

• Genetic background considerations: When using genetically modified mouse models, it's crucial to document the genetic background. For example, one published CARD19-deficient mouse model was backcrossed to C57BL/6 genetic background and confirmed to have a 90:10 C57BL/6J and 129 background through high-throughput genome scanning .

How can RNA sequencing be optimized to identify pathways affected by C9orf89/CARD19 modulation?

RNA sequencing has proven valuable for understanding the global effects of C9orf89/CARD19 modulation. For optimal implementation:

• Sample preparation:

  • Extract total RNA using RNeasy Mini Kit with DNase I treatment

  • Validate RNA quality before sequencing

  • Use appropriate replicates (at least three biological replicates)

• Sequencing parameters:

  • For eukaryotic mRNA libraries, 6 Gb coverage is recommended

  • NovaSeq 6000 or similar high-throughput systems provide adequate read depth

• Data analysis approach:

  • Gene Set Enrichment Analysis (GSEA) is particularly effective for identifying affected pathways

  • Calculate normalized enrichment score (NES), nominal p-value (NOM-p), and false discovery rate q-value (FDR-q)

  • Select categories that are universally up-regulated or down-regulated across replicates

  • Assess relative enrichment of individual genes based on the rank metric score

• Validation of key findings:

  • Perform qPCR for selected genes using SYBR Green

  • Normalize expression to appropriate housekeeping genes (e.g., β-actin)

  • Use volcano plots to visualize differentially expressed genes

A recent study identified 66 categories universally up-regulated in C9orf89 knockout cells, with "cytokine activity" and "cell adhesion mediated by integrin" being particularly relevant to drug resistance mechanisms .

What factors influence antibody selection when studying different isoforms of C9orf89/BinCARD?

When investigating specific isoforms of C9orf89/BinCARD, antibody selection becomes critical due to the existence of multiple isoforms (e.g., BinCARD1 vs. BinCARD2) with different functional implications:

• Epitope consideration: Select antibodies raised against specific regions that can differentiate between isoforms:

  • Some antibodies target AA 1-183 (full-length BinCARD2)

  • Others target specific internal regions (AA 1-100) or epitopes like CTNKEAEKFRNPK

  • The antibody immunogen information is crucial for predicting isoform detection

• Application-specific validation:

  • For western blotting: confirm band size patterns match expected isoform weights

  • For immunohistochemistry: validate using known positive and negative tissues

  • For immunoprecipitation: verify pull-down efficiency with recombinant protein standards

• Cross-reactivity assessment:

  • Test antibodies against both human and mouse/rat samples if studying across species

  • Documented reactivity varies between antibodies, with some showing reactivity to human, mouse, and rat samples, while others are more limited

• Isoform-specific detection strategy:

  • Use RT-PCR with isoform-specific primers in parallel with antibody-based detection

  • Consider absolute quantification using plasmid standards for C9orf89 and AK057716

  • Calculate copy numbers using standard curve equations: Ct = m(log copy number) + b

How does C9orf89 contribute to drug resistance mechanisms in cancer research models?

Recent indirect CRISPR screening with photoconversion has revealed C9orf89 as one of the key genes involved in drug resistance mechanisms:

• Experimental identification:

  • C9orf89 was identified among 39 candidate genes that could reproducibly induce drug resistance in a cell-cell interaction model

  • The screening utilized HEK293T cells and U937 cells in a sophisticated co-culture system with photoconversion tracking

  • Viable U937 colonies under cytarabine exposure were supported by specific mutated HEK293T cells

• Molecular pathway analysis:

  • RNA sequencing of C9orf89-CKO (CRISPR knockout) cells revealed significant pathway alterations

  • Gene set enrichment analysis identified "cytokine activity" and "cell adhesion mediated by integrin" as particularly relevant categories

  • CXCL12 in the "cytokine activity" category was the most commonly enriched gene based on rank metric scores

• Validation methodology:

  • qPCR confirmation of key genes like CXCL12, normalized to β-actin levels

  • Functional validation through drug sensitivity assays

  • Volcano plot visualization of differential gene expression

This research suggests that C9orf89 may play a previously unrecognized role in regulating the tumor microenvironment and mediating drug resistance through cytokine signaling networks and cell adhesion processes.

What autoantibody testing methodologies are most appropriate when studying C9orf89/CARD19 in autoimmune disease models?

When investigating the role of C9orf89/CARD19 in autoimmune disease models, several specialized autoantibody testing methodologies have proven effective:

• Autoantigen microarray profiling:

  • Enables simultaneous measurement of autoantibodies against 124 autoantigens and control proteins

  • Requires serum samples pretreated with DNase-I and diluted 1:50 in PBST buffer

  • Utilizes nitrocellulose film slides with printed autoantigens in duplicates

  • Detection with cy3-labeled anti-mouse IgG and cy5-labeled anti-mouse IgM

  • Scanning via Genepix 4200A scanner with dual laser wavelengths (532 nm and 635 nm)

  • Analysis using Genepix Pro 6.0 software

• ANA (Anti-Nuclear Antibody) testing:

  • HEp-2 cell coated slides provide a standardized substrate for ANA detection

  • Particularly useful in models of systemic lupus erythematosus, where CARD19 deficiency has shown preventive effects

  • Enables visualization of specific nuclear staining patterns associated with different autoimmune conditions

• ELISA-based quantification:

  • Allows for isotype-specific (IgG, IgM, IgA) quantification of autoantibodies

  • Can be used to determine autoantibody titers over time in longitudinal studies

  • Particularly useful for validation of findings from autoantigen arrays

These methodologies, when combined with appropriate controls and CARD19-deficient models, provide comprehensive insights into the role of C9orf89/CARD19 in autoimmune disease pathogenesis and potential therapeutic interventions.

How can inconsistencies in C9orf89 antibody performance across different experimental systems be addressed?

Inconsistencies in C9orf89 antibody performance are commonly reported challenges that can be systematically addressed through:

• Sample preparation optimization:

  • For protein extraction: Use RIPA buffer with protease inhibitors for most applications, but consider specialized mitochondrial isolation buffers for studying native C9orf89 localization

  • For tissue samples: Optimize fixation times (10% formalin for 24-48 hours) and antigen retrieval methods (citrate buffer pH 6.0, EDTA buffer pH 9.0)

  • For cultured cells: Test different lysis conditions to preserve the integrity of C9orf89 protein complexes

• Antibody validation strategies:

  • Test multiple antibodies targeting different epitopes of C9orf89

  • Include positive controls (A549 cells, C2C12 cells, kidney tissue) and negative controls (CRISPR knockout cells if available)

  • Perform peptide competition assays to confirm specificity

  • Use samples with known expression levels for calibration

• Protocol-specific adjustments:

  • For Western blotting: Titrate antibody concentrations (1:1000-1:4000), optimize blocking conditions (5% milk vs. 5% BSA), and test different transfer methods

  • For IHC: Test various antigen retrieval methods and titrate antibody dilutions (1:50-1:100)

  • For ELISA: Optimize coating concentrations, blocking buffers, and detection systems

When implementing these strategies, maintain detailed records of optimization steps to identify which variables most significantly affect antibody performance in your specific experimental system.

What specialized techniques are available for studying C9orf89 interactions with TAK1 and other signaling partners?

Several specialized techniques have proven effective for investigating C9orf89 interactions with TAK1 and other signaling partners:

• Co-immunoprecipitation (Co-IP) optimization:

  • Use antibodies against HA-tag, FLAG-tag, or native proteins (anti-C9orf89, anti-TAK1)

  • Include appropriate controls: rabbit anti-HA (Y-11), rabbit anti-Bcl10 (H-197), rabbit anti-FLAG, mouse anti-FLAG (M2)

  • Validate interactions through reciprocal pulldowns (IP: C9orf89, WB: TAK1 and vice versa)

  • Consider crosslinking approaches for transient interactions

• Signaling pathway analysis:

  • Monitor NF-κB activation through phospho-IκBα (Ser32) detection

  • Assess total IκBα levels as controls

  • Include pathway-specific controls: A20, MCPIP1 (Regnase)

  • Use subcellular fractionation to determine compartment-specific interactions

• Microscopy-based approaches:

  • Utilize fluorescently labeled antibodies (e.g., ALEXA FLUOR® 488 conjugated anti-C9orf89)

  • Perform co-localization studies with mitochondrial markers (e.g., TOMM20)

  • Consider FRET-based approaches for direct protein-protein interaction visualization

  • Include appropriate controls for autofluorescence and non-specific binding

These methodologies, when carefully implemented with appropriate controls, enable detailed characterization of the molecular mechanisms underlying C9orf89/CARD19's regulatory functions in immune signaling pathways.

How can C9orf89 be studied in the context of clinical research and potential therapeutic applications?

Translating C9orf89 research to clinical applications requires specialized approaches that bridge basic science with therapeutic development:

• Biomarker development strategies:

  • Evaluate C9orf89 expression in patient samples using validated antibodies

  • Correlate expression levels with disease progression or treatment response

  • Consider single-cell approaches to identify cell type-specific expression patterns

  • Develop standardized protocols for clinical sample processing and analysis

• Therapeutic targeting approaches:

  • Based on C9orf89's role in BCR signaling and autoimmunity, explore modulation strategies:

    • Small molecule inhibitors of C9orf89-TAK1 interaction

    • Peptide-based approaches targeting specific domains

    • Gene therapy approaches for autoimmune conditions

  • Include appropriate safety assessments given C9orf89's role in normal immune function

• Clinical trial considerations:

  • For inclusion in clinical studies, consider C9orf89 as a biomarker for:

    • Stratifying patients based on expression levels

    • Monitoring treatment response through dynamic changes

    • Predicting autoimmune complications

  • Develop and validate robust assays suitable for clinical laboratory implementation

  • Consider regulatory requirements for companion diagnostic development

While currently at early research stages, C9orf89's demonstrated role in autoimmunity regulation suggests significant potential for therapeutic applications, particularly in B cell-mediated autoimmune disorders and potentially in modulating drug resistance in cancer therapy.