IFIT2 Antibody

What is IFIT2 and what are its primary cellular functions?

IFIT2 (Interferon-induced protein with tetratricopeptide repeats 2) belongs to the IFIT family of proteins that are strongly induced following type I interferon treatment or pattern recognition receptor activation. The protein contains 6 TPR (tetratricopeptide) repeats and has a calculated molecular weight of 56 kDa, though it typically appears at 50-55 kDa on Western blots .

• Binding to viral and cellular mRNAs in AU-rich regions

• Preventing ribosome pausing during translation of bound mRNAs

• Playing a role in inducing apoptosis via the intrinsic mitochondrial pathway involving Bak and Bax proteins

• Potentially functioning as a tumor suppressor in certain cancers, as evidenced by its ability to decrease cell proliferation and increase apoptosis when expressed exogenously in colorectal cancer cells

The protein's function appears to be context-dependent, with surprising pro-viral activities discovered in influenza virus infections despite its canonical antiviral classification.

What applications can IFIT2 antibodies be used for in research settings?

IFIT2 antibodies have been validated for multiple experimental applications, enabling comprehensive study of this protein across different research contexts:

For immunohistochemistry applications, antigen retrieval is recommended using TE buffer at pH 9.0, though citrate buffer at pH 6.0 can serve as an alternative . Optimization of dilutions for each specific experimental system is advised to achieve optimal signal-to-noise ratio.

How can researchers validate IFIT2 antibody specificity?

Validation of IFIT2 antibody specificity should include multiple approaches:

• Knockdown/Knockout Controls: Compare antibody signal in wild-type cells versus IFIT2 knockout or knockdown cells. Published literature shows at least 5 studies utilizing IFIT2 KD/KO approaches for antibody validation .

• Interferon Induction: Since IFIT2 is interferon-inducible, comparing untreated versus interferon-treated samples should show significant upregulation of the protein. This serves as a functional validation of antibody specificity.

• Recombinant Protein Controls: Using purified recombinant IFIT2 as a positive control can confirm antibody specificity at the expected molecular weight.

• Cross-reactivity Testing: When working with human and mouse models, confirm the species-specific reactivity as noted in product information (the referenced antibody shows reactivity with both human and mouse samples) .

• Competing Peptide Assay: Pre-incubating the antibody with the immunogen peptide should abolish specific signal in your application.

What sample preparation methods optimize IFIT2 detection?

For optimal IFIT2 detection across various applications, consider these preparation approaches:

For Western Blotting:

• Lyse cells in RIPA buffer supplemented with protease inhibitors

• Include phosphatase inhibitors if investigating post-translational modifications

• Sonicate samples briefly to shear genomic DNA and reduce viscosity

• Heat samples at 95°C for 5 minutes in reducing sample buffer

For Immunoprecipitation:

• Use gentler lysis buffers (e.g., NP-40 buffer) to preserve protein interactions

• Pre-clear lysates with protein A/G beads to reduce non-specific binding

• Use 0.5-4.0 μg antibody per 1.0-3.0 mg of total protein lysate for optimal precipitation

For Immunohistochemistry:

• Perform antigen retrieval with TE buffer at pH 9.0 (preferred) or citrate buffer at pH 6.0

• Block endogenous peroxidase activity prior to primary antibody incubation

• Optimize incubation time and temperature based on tissue type

For Immunofluorescence:

• Fix cells with 4% paraformaldehyde for 15 minutes

• Permeabilize with 0.1-0.5% Triton X-100

• Use appropriate blocking solutions to minimize background

How should researchers approach investigating IFIT2's dual role in viral infections?

IFIT2 presents a fascinating research case where a canonically antiviral protein can be repurposed as a pro-viral factor specifically during influenza virus infection. To investigate this duality, researchers should consider:

• Viral Strain Specificity: Compare IFIT2's effects across different viral strains. Research has shown that independent selections using different influenza virus strains (WSN and SC35M Flu-GFP) consistently identified IFIT2 as a pro-viral factor, suggesting this is not strain-specific .

• Temporal Expression Analysis: Examine IFIT2 expression and function at different timepoints post-infection. CRISPR/Cas9-knockout selection experiments demonstrated that multi-step IAV replication was reduced by 1-2 logs in IFIT2-deficient cells, indicating stage-specific effects .

• RNA Binding Mutant Comparisons: Utilize the RNA-binding IFIT2 mutant (R292E/K410E, IFIT2ΔRNA) to determine if RNA binding is necessary for pro-viral functions. Research showed that viral gene expression enhancement was lost when using this mutant, indicating RNA binding is crucial for IFIT2's pro-viral activity .

• Host-Pathogen Interaction Studies: Implement techniques that can detect direct IFIT2-viral component interactions, such as cross-linking immunoprecipitation (CLIP) coupled with high-throughput sequencing. This approach revealed that IFIT2 binds to transcripts from all 8 influenza virus genes but not genomic minus-sense vRNA .

• Translation Efficiency Measurements: Employ polysome profiling and ribosome profiling to examine how IFIT2 affects translation of viral versus host mRNAs. Research demonstrated that IFIT2 co-pelleted with ribosomes from infected cell lysate, and this association was disrupted by EDTA treatment that dissociates polysomes .

What methods are most effective for studying IFIT2-RNA interactions?

To investigate IFIT2's RNA binding properties, researchers should consider these methodological approaches:

• CLIP-Seq (Cross-linking Immunoprecipitation followed by Sequencing): This technique provides transcriptome-wide identification of IFIT2 binding sites with nucleotide resolution. Studies have successfully used this approach to demonstrate that IFIT2 binds viral and cellular mRNAs preferentially in AU-rich regions .

• RNA Immunoprecipitation (RIP): For targeted validation of specific RNA interactions, RIP coupled with RT-qPCR has shown ~10-fold enrichment of influenza viral RNAs in IFIT2 immunoprecipitates compared to control .

• Mutational Analysis: Testing the RNA-binding IFIT2 mutant (R292E/K410E) alongside wild-type IFIT2 can confirm the functional importance of RNA binding in experimental systems .

• Capillary Electrophoresis: This technique can be used to analyze RNAs present in IFIT2 RIPs, which has revealed strong enrichment of ribosomal RNAs (rRNAs), including 28S, 18S and 5/5.8S rRNAs in IFIT2 complexes .

• Polysome Association Studies: Polysome pelleting experiments help determine if IFIT2 associates with actively translating ribosomes. Researchers found that IFIT2 co-pelleted with ribosomes from infected cell lysate, and this association was eliminated by EDTA treatment which dissociates polysomes .

When designing experiments to study IFIT2-RNA interactions, researchers should note that background levels of viral sequence can be high in input controls due to influenza virus NP co-migrating with IFIT2 during gel electrophoresis, potentially causing artificial enrichment of viral RNAs in size-matched input controls .

How can researchers properly investigate IFIT2's role in cancer biology?

IFIT2 has shown significant tumor-suppressive properties in colorectal cancer, suggesting important roles in cancer biology. To properly investigate these functions:

• Expression Analysis in Clinical Samples: Compare IFIT2 expression between matched tumor and normal tissues. Studies have found significantly lower IFIT2 expression in colorectal cancer tissues compared to normal tissues .

• Pathway Integration Studies: Investigate IFIT2's relationship with key oncogenic pathways. Research has identified IFIT2 as being down-regulated by Wnt/β-catenin signaling in colorectal cancer cells .

• Reporter Assays: Utilize plasmids containing the 5'-flanking region of IFIT2 to study transcriptional regulation. Dual-luciferase assays revealed that reporter activity was augmented 2.62-fold and 1.98-fold by different β-catenin siRNAs, demonstrating transcriptional repression by β-catenin/TCF7L2 complex .

• Functional Studies:

  • Establish stable cell lines expressing exogenous IFIT2 using retrovirus transduction systems

  • Assess effects on cell proliferation through growth curve analyses

  • Perform cell cycle analysis to quantify sub-G1 populations, which can indicate apoptosis

  • Studies showed that exogenous IFIT2 expression significantly suppressed proliferation of SW480 and HCT116 colorectal cancer cells and increased the sub-G1 population, suggesting induction of apoptosis

• Mechanism Exploration: Investigate whether IFIT2's role in cancer involves its RNA-binding capability, translational regulation functions, or is primarily through its known role in apoptosis induction via the intrinsic mitochondrial pathway involving Bak and Bax proteins .

What are the most reliable approaches to study IFIT2 in the context of interferon responses?

Since IFIT2 is an interferon-stimulated gene, special considerations are needed when studying it in the context of interferon responses:

• Dose-Response Relationships: Establish dose-response curves for different interferon types (α, β, γ) to determine optimal concentrations for IFIT2 induction in your specific cell system.

• Time-Course Experiments: Perform time-course analyses to capture the kinetics of IFIT2 induction, as expression typically peaks between 12-24 hours post-interferon treatment depending on cell type.

• Single-Cell Analysis: Consider using single-cell RNA-seq or single-cell protein analysis techniques to account for cell-to-cell variability in interferon responses, as this heterogeneity can significantly impact experimental outcomes.

• Pathway Inhibition Controls: Include JAK/STAT pathway inhibitors as negative controls to confirm the specificity of IFIT2 induction through canonical interferon signaling.

• Cross-Regulation Assessment: Evaluate how IFIT2 induction might be influenced by other cytokines or stress conditions, as IFITs can be regulated by multiple overlapping pathways.

• Comparative Analysis with Other ISGs: Always include other well-characterized ISGs (e.g., IFIT1, IFIT3, MX1, OAS1) as reference points to contextualize IFIT2 responses, as the research indicates they often show coordinated regulation .

How should researchers interpret contradictory results regarding IFIT2's functions?

When faced with contradictory results about IFIT2's functions (e.g., antiviral vs. pro-viral, pro-apoptotic vs. growth-promoting), consider these approaches:

• Cellular Context Evaluation: The cellular background significantly impacts IFIT2 function. Systematically compare results across different cell types, as IFIT2 may have distinct functions in different cellular environments.

• Virus-Specific Mechanisms: IFIT2's antiviral activity varies depending on the virus, IFIT paralog, and species of origin . When encountering contradictory results, carefully examine the viral systems being studied.

• Binding Partner Analysis: IFIT proteins often function in complexes with other IFITs or host proteins. Perform co-immunoprecipitation studies to identify different binding partners that might explain context-specific functions.

• Post-Translational Modification Profiling: Investigate whether post-translational modifications alter IFIT2 function across different experimental conditions.

• Concentration Dependencies: Assess whether IFIT2 exhibits different functions at different expression levels, as concentration-dependent effects could explain contradictory observations.

• Temporal Dynamics: Evaluate whether the timing of analysis could explain discrepancies, as IFIT2's function may change throughout the course of an infection or cellular response.

For example, when reconciling IFIT2's established antiviral activity with its pro-viral function during influenza infection, researchers discovered that influenza virus specifically repurposes IFIT2's ability to enhance translation efficiency of bound mRNAs—a function that likely evolved to support antiviral responses but is exploited by the virus .

What controls are essential when using IFIT2 antibodies in various applications?

When designing experiments with IFIT2 antibodies, include these essential controls:

For Western Blotting:

• Positive control: Cell lines known to express IFIT2 (A431, RAW 264.7, THP-1 cells)

• Negative control: IFIT2 knockout or knockdown cells

• Loading control: Housekeeping protein to normalize expression levels

• Molecular weight marker: To confirm the expected 50-55 kDa band size

• Interferon-treated samples: As positive controls for IFIT2 induction

For Immunoprecipitation:

• Input sample: To verify protein presence before IP

• IgG control: Same species as the IFIT2 antibody to identify non-specific binding

• Reciprocal IP: If studying interactions, confirm with reverse co-IP

• Pre-clearing control: To assess background binding to beads

For Immunohistochemistry/Immunofluorescence:

• Peptide competition: Pre-incubation with immunizing peptide should abolish signal

• Secondary antibody only: To assess background from secondary antibody

• Known positive tissue: Human skin cancer or kidney tissue have been validated

• Fixation controls: Compare multiple fixation methods if initial results are unclear

How can researchers optimize IFIT2 detection in tissues with low baseline expression?

To detect IFIT2 in tissues with low baseline expression, consider these optimization strategies:

• Signal Amplification Systems:

  • Employ tyramide signal amplification (TSA) for immunohistochemistry

  • Use highly sensitive ECL substrates for Western blotting

  • Consider quantum dot-based detection systems for immunofluorescence

• Sample Preparation Optimization:

  • Test multiple antigen retrieval methods, though TE buffer at pH 9.0 is recommended for IFIT2

  • Extend primary antibody incubation time (overnight at 4°C)

  • Optimize detergent concentration in blocking solutions

• Concentration Techniques:

  • For protein lysates, immunoprecipitate IFIT2 before Western blotting

  • Use larger amounts of starting material when possible

• Pre-induction Approaches:

  • When working with cell lines, pre-treat with type I interferons to upregulate IFIT2 expression

  • For tissue samples, consider using specimens from inflammatory conditions where interferon signaling may be activated

• Detection Method Selection:

  • Chromogenic IHC may provide better sensitivity than fluorescence for tissues with high autofluorescence

  • Consider using fluorochrome-conjugated primary antibodies to eliminate secondary antibody background

What are the most effective strategies for studying IFIT2's translational regulation functions?

To investigate IFIT2's role in translational regulation, researchers should consider these approaches:

• Polysome Profiling: This technique separates mRNAs based on the number of associated ribosomes, allowing assessment of translational efficiency.

  • Compare polysome profiles between wild-type and IFIT2-deficient cells

  • Analyze specific mRNAs across polysome fractions using RT-qPCR

  • Research has shown IFIT2 co-pelleting with ribosomes, with this association disrupted by EDTA treatment

• Ribosome Profiling: This genome-wide approach maps ribosome positions on mRNAs with nucleotide resolution.

  • Can identify ribosome pausing sites that are affected by IFIT2

  • Compare ribosome occupancy patterns between control and IFIT2-expressing cells

  • Studies have shown IFIT2 prevents ribosome pausing on bound mRNAs

• Translation Reporter Assays:

  • Construct reporters containing IFIT2-binding regions (AU-rich sequences)

  • Compare translation efficiency using dual-luciferase reporters

  • Test the effect of the RNA-binding IFIT2 mutant (R292E/K410E) as a negative control

• Metabolic Labeling:

  • Use puromycin incorporation (SUnSET method) to measure global translation rates

  • Perform pulse-labeling with 35S-methionine to quantify newly synthesized proteins

• In Vitro Translation Systems:

  • Reconstitute translation with purified components to directly test IFIT2's effect

  • Compare translation rates and efficiency with and without recombinant IFIT2

When designing these experiments, consider that IFIT2 shows preferential binding to AU-rich regions and appears to enhance translation of bound transcripts, with important implications for both viral replication and cellular responses to infection .