HCFC2 Antibody

What is HCFC2 and what cellular functions does it regulate?

HCFC2 is a member of the host-cell-factor protein family that emerged in early vertebrate evolution through gene duplication. It functions as a critical component of the IRF1 and IRF2 transcriptional machinery that regulates TLR3 and selected interferon-regulated gene (IRG) expression .

Functionally, HCFC2:

• Promotes binding of IRF1 and IRF2 to the Tlr3 promoter

• Is necessary for basal and induced Tlr3 transcription

• Is required for inflammatory cytokine and type I IFN responses to double-stranded RNA

• Enables transcription of a large subset of IRF2-dependent interferon-regulated genes

• Inhibits cell proliferation and activates differentiation-gene expression

The essential nature of HCFC2 is demonstrated by increased susceptibility to viral infections in Hcfc2-deficient mice, particularly to influenza virus and herpes simplex virus 1 infections .

What are the key characteristics of commercially available HCFC2 antibodies?

HCFC2 antibodies are available in multiple formats with varying characteristics:

The observed molecular weight of HCFC2 is approximately 87 kDa (792 amino acids) , although this may vary by species and post-translational modifications.

What are the recommended storage and handling conditions for HCFC2 antibodies?

For optimal antibody performance and longevity:

• Store at -20°C for long-term stability

• Most HCFC2 antibodies are stable for one year after shipment when properly stored

• For certain formulations, aliquoting is unnecessary for -20°C storage

• Typical storage buffer consists of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Some formulations may contain 0.1% BSA for additional stability

These conditions maintain antibody activity while preventing degradation from repeated freeze-thaw cycles.

What controls should be included when using HCFC2 antibodies in flow cytometry and immunoassays?

Proper experimental controls are essential for demonstrating specificity of HCFC2 antibody interactions. Based on established flow cytometry protocols, include these four critical control types:

• Unstained cells control: Addresses false positives due to autofluorescence from endogenous fluorophores

• Negative cell population control: Use cells not expressing HCFC2 to verify target specificity of the primary antibody

• Isotype control: Employ an antibody of the same class as the HCFC2 antibody (typically IgG) but with no specific binding to HCFC2 or related epitopes. This control helps assess undesirable background staining due to Fc receptor binding

• Secondary antibody control: For indirect staining methods, include cells treated with only labeled secondary antibody to measure non-specific binding

Additionally, always use an appropriate blocking agent (typically 10% normal serum) to mask non-specific binding sites. Important: ensure the normal serum is NOT from the same host species as the primary antibody, as this can lead to serious non-specific signals .

How should HCFC2 antibodies be validated before use in critical experiments?

A comprehensive validation approach should include:

• Reactivity verification: Confirm the antibody reacts with your target species. For HCFC2, validated reactivity typically includes human, mouse, and rat samples

• Application-specific testing: Validate in your specific application (WB, IF, IHC, ELISA) using positive control tissues. For HCFC2, mouse testis tissue has been validated as a positive control for Western blot

• Dilution optimization: Determine optimal working dilution; for HCFC2 Western blot applications, starting dilutions typically range from 1:500-1:2000

• Knockout/knockdown validation: When possible, test antibody specificity using HCFC2 knockout or knockdown samples. Research has established HCFC2 knockout models that show a more severe defect than hypomorphic mutations

• Cross-reactivity assessment: Evaluate potential cross-reactivity with related proteins, especially HCFC1, which shares structural similarities with HCFC2

How can ChIP-seq be optimized when studying HCFC2 interactions with transcription factors?

Based on published HCFC2 ChIP-seq protocols that successfully identified HCFC2-IRF2 interactions , consider these optimization strategies:

• Dual cross-linking approach: Implement a two-step cross-linking protocol using:

  • First cross-link with 2 nM EGS for 20 minutes in PBS

  • Follow with 1% formaldehyde for 8 minutes

  • This preserves protein-protein interactions before DNA binding

• Sonication parameters: For optimal chromatin fragmentation:

  • Use a Covaris E220 ultrasonicator or equivalent

  • Parameters: 150 V peak power, duty factor 10, 200 cycles/burst

  • Nine 30-second cycles at 4°C

  • Target fragment size of approximately 150-200 bp

• Antibody selection: Use at least 1 μg of highly specific HCFC2 antibody per 15 μg of soluble chromatin

• Control regions: Include both positive control regions (known HCFC2 binding sites like the Tlr3 IRF-E) and negative control regions (such as Tlr3 intron 3 or Gapdh)

• Data analysis pipeline:

  • Map reads to the genome using Bowtie2

  • Filter for mapping quality scores >10

  • Remove duplicate reads and extend remaining reads to fragment size

  • Normalize to 10 million reads for coverage tracks

  • Identify peaks using MACS with parameters "-p 1e-5 --gsize mm --nomodel True --wig --space=10"

What are the most effective methods for investigating HCFC2-IRF1/IRF2 interactions?

Research has demonstrated several effective approaches for studying these protein interactions:

• Co-immunoprecipitation (Co-IP):

  • Successfully detects HCFC2 interactions with IRF1 and IRF2

  • Use magnetic bead-conjugated antibodies against HCFC2, IRF1, or IRF2

  • Prepare cell lysates in IPH lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, and 0.5% NP-40)

  • Both endogenous and tagged protein approaches have been validated

• Gel shift assays (EMSA):

  • Critical for studying HCFC2's role in facilitating IRF1/IRF2 binding to DNA

  • Use biotinylated oligonucleotide probes containing the IRF-E sequence

  • Mouse Tlr3 IRF-E probe: 5′-TCAGCCTGAAAGTGAAACTTAAGTTGAG-3′

  • Human TLR3 IRF-E probe: 5′-AGCTTTACTTTCACTTTCGAGAGTGC-3′

  • Include competitive assays with 50-fold excess of unlabeled probe

  • For supershift assays, add 2 μg antibodies at room temperature for 15 min after binding reaction

• DNA affinity chromatography pulldown:

  • Detected using 20 fmol biotinylated probe with purified proteins

  • Incubate with streptavidin magnetic beads

  • Wash extensively and analyze by immunoblotting

• Mass spectrometry of protein complexes:

  • Identify HCFC2-interacting partners through immunoprecipitation followed by LC-MS/MS

  • Key identified partners include histone-lysine N-methyltransferase SETD1A, retinoblastoma-binding protein 5 (RBBP5), WD repeat-containing protein 5 (WDR5), and IRF2

How does HCFC2 deficiency affect immune responses to viral infections?

HCFC2 deficiency significantly impairs antiviral immunity through several mechanisms:

• Impaired TLR3 signaling:

  • Reduced TNF production in response to dsRNA (poly(I:C))

  • Decreased phosphorylation of p38, JNK, and ERK

  • Impaired IκB degradation and reduced NF-κB activation

  • Diminished IFN-β production and reduced STAT1 phosphorylation

• Compromised viral defense:

  • Hcfc2-deficient mice show 100% mortality by day 12 after influenza A virus infection

  • Increased susceptibility to HSV-1 infection with development of ataxia and paralysis

  • Reduced serum levels of HSV1-induced IFN-α and IFN-β

  • Defective expression of numerous interferon-regulated genes (IRGs)

• Mechanism of action:

  • HCFC2 forms complexes with IRF1 and IRF2 to facilitate their binding to the Tlr3 IRF-E

  • Without HCFC2, there is reduced association between IRF2 and the IRF-E site in the Tlr3 promoter

  • ChIP-seq identified 365 IRF2 binding sequences enriched in wild-type but not Hcfc2-deficient cells

  • 55.07% of these sequences matched the consensus IRF2-binding site (p=10^-227)

The evidence demonstrates that HCFC2 critically regulates IRF2-dependent transcription of immune response genes necessary for effective viral defense.

What is known about the role of HCFC2 in cell proliferation and differentiation?

Research has revealed that HCFC2 functions as a regulator of cell growth and differentiation:

• Inhibition of cell proliferation:

  • Cells with elevated levels of full-length HCF-2 show slower growth rates

  • HCF-2 expression leads to 4-5 fold increase in cell multinucleation (to ~22% of cells compared to 4% in controls)

  • This phenotype resembles what occurs when HCF-1 function is lost

• Gene expression regulation:

  • Transcriptome analysis revealed that HCF-2 induction leads to:

    • Decreased expression of genes involved in metabolic processes

    • Increased expression of genes related to differentiation and morphogenesis

  • Many of the downregulated genes following HCF-2 induction are also downregulated in cells depleted of HCF-1, suggesting opposing cellular roles

• Developmental relevance:

  • Analysis of RNA-seq databases shows peaks of HCFC2 gene expression during early embryogenesis in fish, frog, and mouse embryos

  • A peak of HCFC2 expression around gastrulation slightly precedes epithelial-mesenchymal transition (EMT)

These findings suggest HCFC2 plays a role in activating differentiation and morphogenesis gene expression programs while inhibiting cellular growth and metabolism.

How can background signals be reduced when using HCFC2 antibodies in immunofluorescence and immunohistochemistry?

When troubleshooting high background in HCFC2 immunostaining:

• Optimize blocking conditions:

  • Use 10% normal serum from the same host species as labeled secondary antibody

  • Ensure the normal serum is NOT from the same host species as the primary antibody

  • Consider alternative blocking agents such as 1-5% BSA or commercial blocking buffers

  • Extend blocking time to 1-2 hours at room temperature

• Antibody dilution optimization:

  • Test serial dilutions of HCFC2 antibody (starting from manufacturer's recommendation)

  • Consider longer incubation at lower concentrations (e.g., overnight at 4°C)

  • For polyclonal antibodies like the rabbit anti-HCFC2, pre-absorption against tissue lysates may help reduce non-specific binding

• Secondary antibody considerations:

  • Use highly cross-adsorbed secondary antibodies to minimize cross-reactivity

  • Include a secondary-only control to assess non-specific binding

  • Consider fluorophores with lower autofluorescence in your tissue type

• Tissue processing improvements:

  • Fresh preparation of fixatives reduces background

  • Optimize fixation time to preserve epitopes while maintaining structure

  • Consider antigen retrieval methods if working with FFPE samples

Why might Western blot detection of HCFC2 show multiple bands or unexpected molecular weights?

Multiple bands or unexpected molecular weights in HCFC2 Western blots may result from:

• Endogenous processing:

  • TLR3 protein shows both endosomally cleaved functional form and uncleaved form (trafficked from ER-Golgi)

  • HCFC2 may undergo similar processing

  • Research has shown that HCFC2^fls^ mutation causes protein instability and degradation, potentially generating multiple fragments

• Post-translational modifications:

  • HCFC2 interacts with several modification enzymes, including histone-lysine N-methyltransferase SETD1A

  • Such interactions suggest HCFC2 may undergo modifications affecting its migration pattern

• Isoforms and splice variants:

  • Different isoforms may exist with tissue-specific expression patterns

  • The expected molecular weight of HCFC2 is 87 kDa (792 amino acids) , but variants may exist

• Technical solutions:

  • Include positive control samples (mouse testis tissue is confirmed positive for HCFC2)

  • Use freshly prepared samples with protease inhibitors

  • Consider gradient gels for better separation

  • Verify antibody specificity using HCFC2 knockout or knockdown samples

How can HCFC2 antibodies be utilized in studying innate immune regulation beyond TLR3 pathways?

Current research suggests several promising applications for HCFC2 antibodies in broader innate immunity studies:

• IRF-regulated gene networks:

  • HCFC2 facilitates IRF1/IRF2 binding to numerous gene targets beyond Tlr3

  • RNA-seq identified 571 genes similarly affected by HCFC2 and IRF2 deficiency

  • After IFN-β treatment, 71% (403 of 571) of these genes remained differentially expressed

  • HCFC2 antibodies could help map the complete network of IRF-regulated genes

• Chromatin modification studies:

  • HCFC2 interacts with chromatin modifiers like SETD1A, RBBP5, and WDR5

  • ChIP-seq approaches using HCFC2 antibodies could reveal how these interactions affect chromatin accessibility

• Viral recognition pathways:

  • Beyond TLR3, HCFC2 may regulate other viral recognition pathways

  • HCFC2 antibodies could help identify whether HCFC2 regulates RIG-I-like receptors or cytosolic DNA sensors

• Interferon-regulated gene expression:

  • HCFC2 deficiency affects expression of key antiviral genes:

    • Trail (TNF-related apoptosis-inducing ligand)

    • Iigp1 (interferon inducible GTPase 1)

    • Mov10 (Moloney leukemia virus 10)

    • Ifi47 (IFN-γ-inducible protein 47)

  • Combined ChIP-seq and RNA-seq approaches could elucidate the mechanisms of this regulation

What is the potential for developing antibodies targeting specific HCFC2 domains for research applications?

The modular structure of HCFC2 offers opportunities for domain-specific antibodies with specialized research applications:

• β-propeller domain antibodies:

  • HCFC2 kelch-like repeats 5 and 6 within the β-propeller domain are minimally required for interaction with IRF2

  • Domain-specific antibodies could selectively disrupt HCFC2-IRF2 interactions

  • Such tools would enable precise dissection of HCFC2 functions

• Customized specificity antibodies:

  • Recent advances in antibody engineering demonstrate computational design of antibodies with customized specificity profiles

  • Biophysics-informed models can identify and disentangle multiple binding modes

  • This approach could generate HCFC2 antibodies with specific high affinity for particular target epitopes

• Cross-species reactive antibodies:

  • HCFC2 is conserved across vertebrates with peaks of expression during early embryogenesis in fish, frog, and mouse

  • Antibodies targeting conserved regions could facilitate evolutionary studies of HCFC2 function

• Nucleolar localization-specific antibodies:

  • HCFC2 shows nucleolar localization patterns that may regulate its functions in differentiation and inhibition of cellular growth

  • Antibodies detecting specific nucleolar-bound conformations could elucidate this regulatory mechanism

These approaches represent the next generation of precision tools for HCFC2 research beyond conventional antibody applications.