C1QTNF4 Antibody

What is C1QTNF4 and what cellular functions has it been shown to regulate?

C1QTNF4 is a member of the C1q/TNF-related protein family that exhibits several important biological functions. Research has shown that C1QTNF4 inhibits vascular smooth muscle cell (VSMC) proliferation and migration by downregulating the FAK/PI3K/AKT pathway, thereby protecting against abnormal neointima formation in blood vessels . Additionally, C1QTNF4 has been genetically linked to systemic lupus erythematosus (SLE), suggesting immunoregulatory functions . In immune contexts, it appears to function primarily within the innate immune system, likely in an anti-inflammatory capacity . Serum C1QTNF4 levels have been found to be decreased in patients with arterial stenosis, suggesting potential biomarker applications .

What receptors mediate C1QTNF4 cellular responses?

Nucleolin has been identified as a cell surface receptor for C1QTNF4 through mass spectrometric analysis . This interaction is mediated specifically by the second C1q-like domain of C1QTNF4 and the C-terminus of nucleolin, particularly within RNA-binding domain 4 and the glycine/arginine rich (GAR) region . Flow cytometry studies have demonstrated that monocytes show high binding affinity for C1QTNF4, correlating with uniform expression of nucleolin on their surface . A subset of B cells (approximately 29% in one study) also bind C1QTNF4, while only a very small percentage of T cells (1-2%) demonstrate binding . Interestingly, C1QTNF4 shows extensive binding to dead or late apoptotic cells, suggesting potential involvement in clearance mechanisms .

What detection methods are available for C1QTNF4 research?

Several validated methodologies are available for C1QTNF4 detection:

When selecting detection methods, researchers should consider the specific research question, sample type, and required sensitivity. For example, ELISA is preferable for quantitative serum analysis, while multiplex immunofluorescence offers advantages for investigating co-localization with cellular markers.

How specific are antibodies against C1QTNF4?

Commercially available and laboratory-generated antibodies against C1QTNF4 show varying degrees of specificity. Monoclonal antibodies generated from hybridoma strains (such as clones 16, 33, and 35) have demonstrated high specificity for C1QTNF4 when validated by Western blot and ELISA techniques . Some polyclonal antibodies developed against the globular domain of human C1QTNF4 can recognize equivalent domains in mouse and rat orthologs without cross-reactivity to related proteins like adiponectin or thioredoxin .

When selecting an antibody, researchers should verify its specificity against closely related C1QTNF family members. Key validation experiments include Western blotting against recombinant proteins, peptide competition assays, and immunoprecipitation followed by mass spectrometry confirmation . For flow cytometry applications, antibodies should be validated using appropriate controls and isotype-matched antibodies .

What experimental protocols are recommended for studying C1QTNF4-nucleolin interactions?

The interaction between C1QTNF4 and nucleolin can be investigated through several complementary approaches:

Co-immunoprecipitation (Co-IP):

• Express GFP-tagged C1QTNF4 in appropriate cell lines (e.g., SK-N-AS neuroblastoma cells)

• Prepare cell lysates under non-denaturing conditions

• Perform immunoprecipitation using GFP-Trap or anti-GFP antibodies

• Analyze precipitates by SDS-PAGE and immunoblot with anti-nucleolin antibodies

• Confirm specificity through mass spectrometry identification

Competition binding assays:

• Express recombinant nucleolin domain constructs (R1234G, R4G, R123) in E. coli

• Incubate cells with fluorescently-labeled C1QTNF4 with or without nucleolin domain constructs

• Measure competition for binding using flow cytometry

• Alternative approach: Use the nucleolin-targeting DNA aptamer AS1411 as a competitive inhibitor

Internalization studies:

• Incubate target cells (monocytes) with fluorescently-labeled C1QTNF4

• Analyze internalization by imaging flow cytometry at various time points

• Visualize subcellular trafficking using confocal microscopy with appropriate markers for endocytic compartments

These approaches should be complemented with appropriate controls, including nucleolin knockdown experiments to confirm specificity of the observed interactions.

How can I optimize immunofluorescence staining for C1QTNF4 in vascular tissues?

Optimizing immunofluorescence staining for C1QTNF4 in vascular tissues requires careful attention to several technical factors:

Tissue preparation protocol:

• Fix tissue samples in 4% paraformaldehyde (optimal fixation time depends on tissue thickness)

• Process tissues for paraffin embedding or freeze in OCT compound

• Section at 5-8 μm thickness

• For paraffin sections: deparaffinize, rehydrate, and perform antigen retrieval (citrate buffer, pH 6.0)

• For frozen sections: fix briefly in acetone or 4% PFA after sectioning

Antibody optimization:

• Test multiple antibody dilutions (typically 1:100-1:500) to determine optimal signal-to-noise ratio

• Include appropriate blocking (5-10% normal serum from secondary antibody species plus 0.1-0.3% Triton X-100)

• Optimize primary antibody incubation (overnight at 4°C is typically effective)

• Use fluorophore-conjugated secondary antibodies appropriate for your imaging system

Critical controls:

• Omit primary antibody (secondary antibody only)

• Use isotype-matched control antibodies

• Include known positive tissue samples

• Consider using C1QTNF4-knockout tissues as negative controls if available

For vascular-specific analyses, co-stain with VSMC markers (α-smooth muscle actin) to confirm co-localization in the vascular wall . When studying immune cell interactions, consider multiplex staining with monocyte or B-cell markers to identify specific cellular targets .

What signaling pathways are modulated by C1QTNF4 and how can they be investigated?

C1QTNF4 modulates several signaling pathways that can be investigated using the following approaches:

FAK/PI3K/AKT Pathway Investigation:

• Treat VSMCs with recombinant C1QTNF4 at various concentrations (50-200 ng/mL)

• Harvest cells at different time points (0-60 minutes for immediate signaling, 2-24 hours for downstream effects)

• Analyze phosphorylation states by Western blotting using phospho-specific antibodies:

  • Phospho-FAK (Y397)

  • Phospho-PI3K p85 (Y458)

  • Phospho-AKT (S473 and T308)

• Confirm pathway specificity using pharmacological inhibitors (e.g., FAK inhibitor PF-573228, PI3K inhibitor LY294002, AKT inhibitor MK-2206)

Transcriptomic Approach:

• Perform RNA-seq analysis on tissues from control versus C1QTNF4-treated or C1QTNF4-knockout models

• Validate candidate genes by RT-qPCR

• Conduct pathway enrichment analysis to identify broader signaling networks

IL-6 Signaling Investigation:

• Measure IL-6 expression by ELISA and qPCR in response to C1QTNF4 treatment

• Analyze JAK/STAT pathway activation through phospho-STAT3 detection

• Use siRNA knockdown of pathway components to confirm their role in C1QTNF4-mediated effects

An integrated approach combining these methods will provide comprehensive understanding of how C1QTNF4 influences cellular signaling networks and downstream functional outcomes.

What animal models are most effective for studying C1QTNF4 function in vascular diseases?

Several validated animal models have proven effective for investigating C1QTNF4 function in vascular disease contexts:

Genetically Modified Mouse Models:

• C1QTNF4-transgenic mice: Overexpress C1QTNF4 to study protective effects against vascular injury

• C1QTNF4^−/−^ knockout mice: Allow investigation of loss-of-function phenotypes

• AAV9-mediated VSMC-specific C1QTNF4 restoration in knockout mice: Enables tissue-specific rescue experiments

Vascular Injury Models:

• Wire injury model (mice): Insert guidewire into femoral artery to induce mechanical endothelial denudation and subsequent neointimal formation

• Balloon injury model (rats): Use balloon catheter to create controlled vascular damage

• Analysis timepoints: Typically 7, 14, and 28 days post-injury to capture different phases of vascular remodeling

Analytical Approaches:

• Histomorphometry: Measure intima/media ratios, neointimal area, and luminal stenosis

• Immunohistochemistry: Analyze VSMC proliferation (Ki67), phenotypic markers, and inflammatory infiltrates

• Molecular profiling: Perform RT-qPCR and Western blotting on vessel segments

• Functional assessment: Evaluate vascular reactivity using wire myography

How can I distinguish between C1QTNF4 and other structurally related proteins?

Differentiating C1QTNF4 from other structurally related proteins requires careful experimental design and validation:

Antibody Selection and Validation:

• Choose antibodies targeting unique epitopes in C1QTNF4 not conserved in other family members

• Validate specificity by Western blotting against recombinant proteins from multiple C1QTNF family members

• Perform peptide competition assays to confirm epitope specificity

• Verify the absence of cross-reactivity with adiponectin, which shares structural features with C1QTNF proteins

Molecular Characteristics:

• Exploit size differences: C1QTNF4 migrates at a distinct molecular weight compared to other family members

• Target the second C1q-like domain, which is structurally unique in C1QTNF4

• Design primers or probes targeting non-conserved regions for RT-PCR or in situ hybridization

Functional Discrimination:

• Leverage C1QTNF4's specific binding to nucleolin, which may not be shared by other family members

• Utilize cellular specificity: C1QTNF4 preferentially binds monocytes and subsets of B cells

• Examine specific signaling pathway effects, such as FAK/PI3K/AKT modulation in VSMCs

For definitive identification, consider using mass spectrometry-based approaches that can identify unique peptide sequences specific to C1QTNF4, providing unambiguous discrimination from other family members.

What challenges exist in developing and validating C1QTNF4 antibodies for research applications?

Researchers developing and validating C1QTNF4 antibodies face several significant challenges:

Production Challenges:

• Protein aggregation: Full-length human C1QTNF4 tends to form aggregates in expression systems, complicating antigen preparation

• Domain-specific considerations: The globular domain may provide better antigens than full-length protein

• Post-translational modifications may differ between recombinant and native C1QTNF4

Validation Requirements:

• Specificity testing against other C1QTNF family members is essential to prevent cross-reactivity

• Testing across multiple applications (Western blot, ELISA, immunofluorescence, flow cytometry) is necessary as antibodies may perform differently in various contexts

• Cross-species reactivity should be thoroughly evaluated when antibodies will be used in animal models

Application-Specific Validation:

• For immunohistochemistry/immunofluorescence: Validate staining patterns with knockout controls

• For flow cytometry: Confirm specific binding to known C1QTNF4-responsive cells (monocytes, B cells)

• For Western blotting: Verify detection of both recombinant and endogenous protein at the correct molecular weight

When generating monoclonal antibodies, hybridoma screening should include multiple validation methods to ensure selected clones perform well across all intended applications . Extensive characterization is essential before employing antibodies in complex experimental systems.