ABCF1 Antibody

What is ABCF1 and why is it significant for immunological research?

ABCF1 is a unique member of the ATP Binding Cassette (ABC) transporter family that lacks transmembrane domains, distinguishing it from other ABC proteins. Its significance stems from its role as a cytosolic nucleic acid sensor that regulates CXCL10 production, interferon-β expression, and downstream type I interferon responses in various cell types . ABCF1 is expressed in human airway epithelial cells and functions in modulating innate immune activities, including Toll-like receptor (TLR) signaling pathways, making it relevant for research on respiratory viral infections and inflammatory responses .

What are the cellular localization patterns of ABCF1?

ABCF1 has multiple subcellular localizations, being found in the cytoplasm, cytosol, nuclear envelope, and nucleoplasm . This diverse distribution reflects its multifunctional nature in cellular processes. When conducting immunofluorescence or immunohistochemistry experiments, researchers should expect to observe signal in these various compartments, with potentially different intensities depending on cell type and activation state. This localization pattern is important for experimental design and interpretation when using ABCF1 antibodies for cellular imaging.

What techniques can be used to verify ABCF1 antibody specificity?

To verify ABCF1 antibody specificity, researchers should implement multiple validation approaches:

• Western blot analysis using positive control samples (such as HeLa or HepG2 cell lysates) to confirm the expected molecular weight of approximately 120 kDa (observed) versus the calculated 96 kDa

• siRNA knockdown experiments to demonstrate reduced signal with ABCF1 depletion

• Immunoprecipitation followed by mass spectrometry to confirm target binding

• Comparing results across multiple antibodies targeting different epitopes of ABCF1

• Including appropriate negative controls such as isotype control antibodies or secondary-only controls

These validation steps are essential before proceeding with functional studies to ensure experimental rigor and reproducibility.

How should ABCF1 expression be assessed in human airway epithelial samples?

For human airway epithelial samples, a multi-modal approach is recommended:

• In situ hybridization: Using RNAscope™ probes targeting ABCF1 (such as construct targeting 1713-2726 of NM_001025091.1) to detect gene expression in tissue sections

• Immunohistochemistry: Using anti-ABCF1 antibodies (e.g., HPA017578, Sigma-Aldrich) at 1:100 dilution in 3% Casein in TBST buffer

• Immunoblotting: For protein quantification, using Mini-Protean TGX stain-free gels with anti-ABCF1 antibody (1:100) and anti-rabbit HRP-linked secondary antibody (1:2000)

• qRT-PCR: For transcript level quantification in isolated cells

This comprehensive approach provides corroborating evidence of both gene and protein expression, which is critical when studying ABCF1 in primary human samples where expression levels may vary between individuals.

What are the optimal conditions for Western blot detection of ABCF1?

For optimal Western blot detection of ABCF1:

The discrepancy between observed (120 kDa) and calculated (96 kDa) molecular weights may be due to post-translational modifications, which should be considered when interpreting results .

How does ABCF1 function as a dsDNA sensor in antiviral responses?

ABCF1 functions as a cytosolic nucleic acid sensor specifically recognizing dsDNA motifs, including those from pathogens like Listeria monocytogenes and HIV . Methodologically, this function can be studied by:

• Transfecting human airway epithelial cells with dsDNA mimics such as interferon stimulatory DNA (ISD) or VACV-70 at concentrations of 0.1-3.16 μg/ml

• Measuring downstream production of CXCL10 and type I interferons via ELISA or qPCR

• Comparing responses in ABCF1-sufficient versus ABCF1-knockdown cells (using siRNA approaches targeting ABCF1 transcript)

• Analyzing IRF-3 phosphorylation and nuclear translocation through Western blotting and immunofluorescence

Research shows that ABCF1 knockdown attenuates the expression of genes involved in antiviral responses following dsDNA challenge, confirming its role in nucleic acid sensing pathways .

What methods can be used to investigate ABCF1's interaction with the OAS1-RNaseL pathway?

To investigate ABCF1's interaction with the OAS1-RNaseL pathway:

• Co-immunoprecipitation: Using ABCF1 antibodies to pull down protein complexes, followed by Western blotting for OAS1 and ABCE1

• Proximity ligation assay (PLA): To visualize protein-protein interactions in situ between ABCF1 and OAS1

• siRNA knockdown: Depleting ABCF1 and measuring changes in OAS1 activity and RNaseL activation using specific RNA degradation assays

• Poly(I:C) stimulation: Treating cells with poly(I:C) (1 μg/ml for 24h) to activate dsRNA pathways and analyzing phosphorylation of ISG proteins in ABCF1-depleted versus control cells

• Viral challenge assays: Using VSV or SARS-CoV-2 infection models to assess functional outcomes of ABCF1-OAS1 interaction

These approaches help elucidate the unexpected role of ABCF1 in linking innate and adaptive immunity through its interaction with OAS1 and subsequent modulation of RNaseL activity .

How does ABCF1 connect innate and adaptive immune responses?

Research reveals that ABCF1 functions as a crucial link between innate and adaptive immunity, with several research methodologies to investigate this connection:

• Flow cytometry analysis: Using ABCF1 heterozygous mouse models to assess CD4+ and CD8+ T cell populations in spleen and thymus

• Tetramer staining: Evaluating virus-specific CTL responses in ABCF1+/- mice compared to wild-type controls

• IFNγ production assays: Measuring functional T cell responses through cytokine production

• CTL assay: Assessing cytotoxic T lymphocyte function in ABCF1-deficient models

Experimental evidence indicates that ABCF1+/- mice produce fewer functional cytotoxic T lymphocytes, with a significant reduction in CD8+ T cells after immune challenge . This suggests ABCF1 plays a regulatory role in T cell maturation processes, particularly affecting CD8+ T lymphocyte development and function.

What are the key considerations when using ABCF1 antibodies for immunohistochemistry in patient samples?

When performing immunohistochemistry with ABCF1 antibodies on patient samples:

• Fixation protocol: Use 10% neutral buffered formalin fixation for 24-48 hours to preserve epitope integrity

• Antigen retrieval: Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) to optimize antibody binding

• Antibody dilution: Use at 1:50 - 1:200 dilution as recommended

• Controls: Include positive controls (known ABCF1-expressing tissues), negative controls (isotype antibody), and ABCF1-depleted samples when possible

• Scoring system: Develop a standardized scoring system for ABCF1 expression (intensity and percentage of positive cells)

• Multi-antibody panels: Consider using multi-antibody panels to co-localize ABCF1 with markers of immune cell subsets or activation states

Patient-derived samples require careful standardization and validation to account for biological variability and to ensure reproducible results across different clinical specimens.

How can researchers overcome non-specific binding issues with ABCF1 antibodies?

Non-specific binding is a common challenge with antibodies. For ABCF1 antibodies:

• Optimize blocking: Extend blocking time to 2 hours using 5% BSA or non-fat milk in TBST

• Adjust antibody concentration: Titrate the primary antibody to determine optimal concentration that maximizes specific signal while minimizing background

• Increase washing duration: Perform 5-6 washes of 10 minutes each with TBST

• Use alternative antibodies: Compare results with antibodies targeting different epitopes of ABCF1

• Pre-adsorption: Pre-incubate antibody with the immunizing peptide to confirm specificity

• Use lysates from ABCF1 knockdown cells: As negative controls to identify non-specific bands

For immunohistochemistry specifically, additional steps may include using biotin-avidin blocking kits if using biotin-based detection systems, and implementing Sudan Black B treatment to reduce autofluorescence in tissue sections.

What strategies can improve detection of ABCF1 in low-expressing samples?

For low-expressing samples, enhance ABCF1 detection through:

• Signal amplification techniques: Use tyramide signal amplification or polymer-based detection systems

• Increased sample concentration: Load more protein (50-80 μg) for Western blot applications

• Longer exposure times: For chemiluminescent detection, use incremental exposure times

• Enrichment approaches: Perform immunoprecipitation before Western blotting

• More sensitive detection methods: Use fluorescent secondary antibodies with digital imaging systems

• RNA detection: Complement protein detection with more sensitive RT-qPCR or RNA-scope approaches

• Immunoprecipitation-mass spectrometry: For definitive identification in complex samples

These approaches can help overcome detection limitations when studying ABCF1 in samples with naturally low expression levels or following experimental manipulations.

How might ABCF1 antibodies be used to study its role in disease pathogenesis?

ABCF1 antibodies can be employed in several experimental approaches to investigate disease associations:

• Tissue microarrays: Compare ABCF1 expression across healthy versus diseased tissues (cancer, neurodegenerative disorders)

• Patient-derived xenografts: Evaluate ABCF1 expression and localization in animal models bearing patient tumors

• Single-cell techniques: Couple ABCF1 antibodies with single-cell sequencing approaches to identify cell populations with altered ABCF1 expression

• Proximity proteomics: Use ABCF1 antibodies for BioID or APEX2 approaches to identify disease-specific interaction partners

• Phospho-specific antibodies: Develop and employ antibodies recognizing post-translationally modified forms of ABCF1

Research has implicated ABCF1 dysregulation in various diseases including cancer and neurodegenerative disorders , making these approaches valuable for understanding its pathophysiological roles and potential as a therapeutic target.

What methods can be used to study ABCF1's role in TLR signaling pathways?

To investigate ABCF1's involvement in TLR signaling:

• Stimulation experiments: Treat cells with specific TLR ligands (e.g., LPS for TLR4) with and without ABCF1 knockdown

• Phosphorylation analysis: Monitor phosphorylation of downstream signaling molecules (e.g., NF-κB, IRF3) through Western blotting

• Ubiquitination studies: Analyze K63-polyubiquitination patterns influenced by ABCF1, as it has been shown to target key proteins for this post-translational modification

• TLR trafficking assays: Monitor TLR4 endocytosis in the presence or absence of ABCF1 using flow cytometry or microscopy

• Reporter assays: Utilize NF-κB or ISRE reporter constructs to measure pathway activation

• Transcriptomics: Perform RNA-seq analysis on ABCF1-deficient cells after TLR stimulation to identify differentially regulated gene networks

Gene Ontology analyses have revealed significant interactions between ABCF1 and TLR signaling, suggesting it plays a multifactorial role in innate immunity regulation in human airway epithelial cells .