IFITM1 Antibody

What is IFITM1 and what biological role does it play?

IFITM1 (Interferon-Induced Transmembrane Protein 1) is a member of the IFITM family of proteins that function as important host effector molecules in the type I interferon response against viruses. It is primarily expressed following interferon stimulation and acts as a potent antiviral effector against multiple viruses, including hepatitis C virus (HCV). IFITM1 contains two hydrophobic membrane-associated domains (M1 and M2) separated by a conserved intracellular loop (CIL), with distinct N- and C-terminal domains that differentiate it from other IFITM family members . The protein is part of a coordinated cellular immune response that restricts viral infection at early stages, particularly during viral entry. The promoter regions of IFITM genes contain Interferon-Stimulated Response Elements (ISRE) and Gamma-Activated Sequences (GAS), allowing rapid upregulation following interferon signaling .

What is the cellular localization pattern of IFITM1 compared to other IFITM proteins?

IFITM1 demonstrates a distinct localization pattern compared to other family members. While IFITM2 and IFITM3 are predominantly localized to intracellular compartments within the cytoplasm, IFITM1 is mainly distributed at the plasma membrane with some intracellular presence . Immunofluorescence studies using Na⁺/K⁺-ATPase (a plasma membrane biomarker) have confirmed that IFITM1 shows strong colocalization with the plasma membrane, while IFITM2 and IFITM3 appear to reside primarily in cytoplasmic compartments . This distinct localization pattern is crucial for understanding the differential antiviral mechanisms of these proteins, as IFITM1's surface expression allows it to interact directly with viral entry receptors such as CD81 .

What is the membrane topology of IFITM1?

IFITM1 adopts a specific topological orientation in the plasma membrane where the N-terminus points into the cytoplasm and the C-terminus extends extracellularly . This topology has been experimentally verified using IFITM1 constructs with dual tags (HA at the N-terminus and Myc at the C-terminus). When plasma membranes are intact, the N-terminal HA tag cannot be detected by antibodies, but becomes accessible when membranes are permeabilized, confirming that the N-terminus faces the cytoplasm . This topological information is critical for designing appropriate antibodies for different experimental applications and understanding how IFITM1 interacts with viral components during infection.

What are the recommended applications for IFITM1 antibodies in viral research?

IFITM1 antibodies can be effectively employed in multiple research applications to investigate viral restriction mechanisms. Western blotting can quantify IFITM1 expression levels following interferon treatment or viral infection, while immunohistochemistry on paraffin-embedded sections allows visualization of tissue-specific expression patterns . For studying subcellular localization, immunofluorescence microscopy with IFITM1 antibodies can reveal the distribution pattern and potential colocalization with viral entry factors like CD81 . When designing experiments, researchers should select antibodies that target specific regions (N-terminal or C-terminal) depending on the protein's orientation and accessibility in their experimental system. For functional studies, combining antibody-based detection methods with virological assays such as HCVpp (HCV pseudoparticle) entry assays can establish correlations between IFITM1 expression patterns and antiviral activity .

How do I validate the specificity of an IFITM1 antibody for my research?

Validating IFITM1 antibody specificity requires a multi-step approach due to the high sequence homology between IFITM family members. Start with positive and negative controls: use cells treated with type I interferons (which upregulate IFITM1) as positive controls and IFITM1 knockdown cells (using specific shRNA) as negative controls . Western blot analysis should show a single band at the expected molecular weight (approximately 17 kDa), with increased intensity in interferon-treated samples and decreased signal in knockdown cells. Cross-reactivity with IFITM2 and IFITM3 should be assessed using overexpression systems of each individual protein. For immunofluorescence applications, verify specificity by examining cellular localization patterns—IFITM1 should predominantly show plasma membrane staining, unlike the intracellular patterns of IFITM2/3 . Additionally, peptide competition assays using the immunizing peptide (derived from the N-terminal region of human IFITM1) can confirm binding specificity .

What are the important considerations when choosing between N-terminal and C-terminal IFITM1 antibodies?

The choice between N-terminal and C-terminal targeting antibodies should be guided by IFITM1's membrane topology and your experimental design. Since IFITM1's N-terminus faces the cytoplasm while the C-terminus extends extracellularly , this orientation affects epitope accessibility in different applications:

• For immunofluorescence on non-permeabilized cells: C-terminal antibodies are preferred as they can access the extracellular epitope

• For co-immunoprecipitation of cytoplasmic interaction partners: N-terminal antibodies may be more effective

• For detecting post-translational modifications: Select antibodies that don't recognize regions containing known modification sites (such as palmitoylation sites in the CIL domain)

• For detecting protein-protein interactions: Consider which domain interacts with your protein of interest

Additionally, N-terminal antibodies might better distinguish IFITM1 from IFITM2/3, as IFITM1 lacks the 20-21 amino acid N-terminal extension present in the other family members . When studying membrane-associated complexes, consider whether your antibody might disrupt important protein-protein interactions, particularly as IFITM1 is known to interact with viral entry factors like CD81 .

How can I use IFITM1 antibodies to investigate virus-specific restriction mechanisms?

To investigate virus-specific restriction mechanisms, implement a systematic experimental approach utilizing IFITM1 antibodies. Begin with establishing stable cell lines expressing tagged IFITM1 (e.g., FLAG-tagged) to facilitate detection with high specificity . For viral entry studies, compare HCV pseudoparticle (HCVpp) entry rates between control and IFITM1-expressing cells using luciferase reporter assays, while confirming IFITM1 expression levels via western blotting with antibodies . Fluorescence microscopy combining IFITM1 antibodies with markers for viral entry factors (such as CD81 for HCV) can reveal potential interaction sites. Advanced techniques including Fluorescence Resonance Energy Transfer (FRET) and Proximity Ligation Assays (PLA) can definitively demonstrate physical interactions between IFITM1 and viral components or entry factors . For endosomal escape inhibition studies, employ electron microscopy to visualize viral particles trapped in endosomal compartments in IFITM1-expressing cells . When analyzing different viruses, use virus-specific reporter systems alongside consistent IFITM1 detection methods to enable direct comparison of restriction mechanisms across viral families.

What experimental approaches can resolve contradictory data on IFITM1 function in different viral systems?

Resolving contradictory data on IFITM1 function across viral systems requires a comprehensive experimental strategy addressing multiple variables. First, standardize cell models by establishing stable cell lines expressing equivalent levels of IFITM1 across experiments, verified by quantitative western blotting with calibrated antibody concentrations . Consider cell-type specific factors by comparing IFITM1 localization and function in relevant primary cells versus cell lines, using immunofluorescence to document potential differences in distribution patterns . Systematically investigate post-translational modifications by examining IFITM1 palmitoylation status across cell types and viral infections, as S-palmitoylation is essential for antiviral function . Implement single-cell analysis techniques that combine antibody-based IFITM1 detection with viral infection markers to distinguish cell-to-cell variability from population effects. For time-dependent effects, conduct time-course experiments with synchronized infections and fixed timepoint antibody detection to identify whether contradictions result from temporal dynamics rather than absolute differences. Finally, create site-specific mutants of IFITM1 to identify whether functional residues show virus-specific importance, particularly focusing on the conserved cysteine residues in the CIL domain and the membrane-associated domains .

How do post-translational modifications affect IFITM1 detection with antibodies?

Post-translational modifications (PTMs) can significantly impact IFITM1 detection with antibodies through several mechanisms. S-palmitoylation of conserved cysteine residues, particularly in the conserved intracellular loop (CIL), can alter antibody binding if the epitope includes or is adjacent to these modification sites . This palmitoylation is critical for IFITM1's antiviral function and membrane association, and modified IFITM1 may display altered mobility on SDS-PAGE, appearing at a higher apparent molecular weight than predicted. When using antibodies targeting regions containing PTM sites, consider treating samples with depalmitoylating agents (like hydroxylamine) in parallel experiments to determine whether modification status affects detection sensitivity . Similarly, tyrosine phosphorylation, particularly at conserved tyrosine residues, can interfere with antibody recognition if the phosphorylated residue is within the epitope. For comprehensive analysis, researchers should compare results from antibodies targeting different regions of IFITM1 and consider using phospho-specific antibodies when investigating signaling-dependent regulation. When developing quantitative assays, validate whether your antibody's binding affinity is affected by the modification status of IFITM1 under different cellular conditions, particularly following interferon stimulation which can alter both expression levels and modification patterns .

What are the optimal fixation and permeabilization methods for IFITM1 immunofluorescence studies?

The optimal fixation and permeabilization protocol for IFITM1 immunofluorescence depends on which aspects of IFITM1 biology you're investigating. For plasma membrane localization studies, use 4% paraformaldehyde fixation for 10-15 minutes at room temperature to preserve membrane structure, followed by selective permeabilization experiments: perform parallel staining with and without permeabilization to differentiate between membrane-associated and intracellular pools of IFITM1 . For detecting the topological orientation, use dual-tagged IFITM1 constructs (e.g., HA-IFITM1-Myc) and compare antibody accessibility under permeabilized versus non-permeabilized conditions to confirm that the N-terminus faces the cytoplasm while the C-terminus extends extracellularly . When studying colocalization with membrane proteins like Na⁺/K⁺-ATPase, mild detergent permeabilization with 0.1% Triton X-100 for 5-10 minutes preserves membrane structure while allowing antibody access . For endosomal localization studies comparing IFITM1 with IFITM2/3, methanol fixation at -20°C for 10 minutes may better preserve intracellular compartments. Always validate your fixation method by comparing staining patterns with live-cell imaging of fluorescently-tagged IFITM1 to ensure the fixation process doesn't artificially alter localization patterns.

How can I design experiments to distinguish between direct and indirect antiviral effects of IFITM1?

Designing experiments to distinguish direct from indirect antiviral effects of IFITM1 requires controlling for multiple variables and using complementary approaches. Implement time-of-addition experiments where IFITM1 expression is induced at different stages of the viral life cycle using inducible expression systems, then detect both IFITM1 (using validated antibodies) and viral markers to determine stage-specific inhibition . Use pseudotyped virus particles (like HCVpp) that complete only the entry step of the viral life cycle to isolate entry-specific effects, combined with immunofluorescence to correlate IFITM1 localization with restricted viral entry . For direct interaction studies, employ co-immunoprecipitation with IFITM1 antibodies followed by detection of viral proteins, or use advanced techniques like FRET and PLA to demonstrate physical proximity between IFITM1 and viral components . Create chimeric IFITM proteins by swapping domains between IFITM1 and non-restrictive membrane proteins, then use immunofluorescence to correlate localization patterns with antiviral activity to identify essential functional domains. Control for indirect effects by analyzing the impact of IFITM1 expression on general cellular processes like endosomal acidification using pH-sensitive dyes alongside immunofluorescence detection of IFITM1 . Finally, conduct electron microscopy studies to visualize the physical relationship between IFITM1-positive membranes and viral particles during infection, particularly focusing on endosomal escape events .

What controls should be included when using IFITM1 antibodies in virus restriction studies?

A comprehensive control strategy is essential when using IFITM1 antibodies in virus restriction studies. Include these key controls:

When analyzing results, always quantify both IFITM1 expression levels and viral restriction phenotypes to establish dose-response relationships and account for cell-to-cell variability in expression.

How should researchers interpret changes in IFITM1 localization following viral infection?

When interpreting changes in IFITM1 localization following viral infection, researchers should consider several critical factors. First, establish a clear baseline by documenting the steady-state distribution of IFITM1 in uninfected cells using immunofluorescence, noting that IFITM1 normally exhibits both plasma membrane and some intracellular localization . Following infection, quantify changes in distribution patterns using co-localization analysis with markers for different cellular compartments (plasma membrane, early endosomes, late endosomes, and lysosomes). Relocalization to endosomal compartments may indicate recruitment to sites of viral entry and membrane fusion, supporting a direct antiviral mechanism . Compare the timing of relocalization with known kinetics of viral entry to determine whether changes are preventative or responsive. For accurate interpretation, use time-course experiments to distinguish between redistribution of existing IFITM1 versus synthesis and trafficking of new protein following infection. Consider whether localization changes correlate with altered post-translational modifications, particularly S-palmitoylation which affects membrane association and antiviral function . Also evaluate whether relocalization patterns differ between restrictive and permissive cell types, which may explain cell-type specific differences in antiviral activity. To determine functional significance, create IFITM1 mutants with altered trafficking patterns and assess their antiviral activity compared to wild-type protein.

What analytical approaches can distinguish between expression level effects and functional changes in IFITM1 antiviral activity?

Distinguishing between expression level effects and functional changes in IFITM1 antiviral activity requires sophisticated analytical approaches. Implement dose-response analysis by creating stable cell lines expressing IFITM1 at different controlled levels, then plot viral restriction against protein expression quantified by calibrated western blotting to establish the relationship between abundance and function . Use flow cytometry with dual staining for IFITM1 and viral antigens at the single-cell level to correlate expression with protection, allowing identification of potential threshold effects versus proportional protection. For functional analysis independent of expression level, create cell lines expressing equivalent amounts of wild-type versus mutant IFITM1 (verified by quantitative western blotting), then compare their antiviral activities to identify mutations that specifically affect function without altering expression . Employ biochemical fractionation to determine the proportion of IFITM1 in different cellular compartments, as the functional pool may be a subset of the total detected protein. When analyzing post-translational modifications, calculate modification stoichiometry (proportion of IFITM1 molecules modified) rather than just detecting presence/absence. For mathematical modeling of expression versus function, use partial least squares regression analysis to separate the contributions of expression level, localization pattern, and modification status to the observed antiviral phenotype.

How do I troubleshoot inconsistent IFITM1 antibody staining patterns across experiments?

Inconsistent IFITM1 antibody staining patterns can result from multiple technical and biological variables that must be systematically addressed. Create a standardized protocol with consistent fixation parameters (agent, concentration, duration, and temperature), as overfixation can mask epitopes while underfixation may allow protein redistribution . Optimize antibody dilution by performing titration experiments for each new lot of antibody, as even antibodies from the same manufacturer may vary in optimal working concentration between batches . For membrane proteins like IFITM1, permeabilization conditions significantly impact staining patterns; compare different detergents (Triton X-100, saponin, digitonin) at various concentrations to identify optimal conditions that preserve membrane structure while allowing antibody access . Control for cell cycle and density effects by standardizing seeding density and synchronizing cultures, as IFITM1 localization may vary with these parameters. For interferon-induced expression, establish a consistent protocol for timing and concentration of interferon treatment relative to staining . If using multiple fluorophore-conjugated secondary antibodies, verify there's no bleed-through between channels and that secondary antibodies don't exhibit non-specific binding. Finally, consider epitope masking due to protein-protein interactions or post-translational modifications by comparing different antibodies targeting distinct regions of IFITM1 . Document and standardize image acquisition parameters including exposure time, gain, and post-processing steps to ensure quantitative comparisons between experiments.

What methodological advances are needed to better understand IFITM1 membrane topology and dynamics?

Advancing our understanding of IFITM1 membrane topology and dynamics requires innovative methodological approaches. Super-resolution microscopy techniques (STORM, PALM) coupled with specific IFITM1 antibodies could reveal nanoscale organization within the plasma membrane and potential clustering during viral challenge that conventional microscopy cannot detect . Live-cell imaging using split fluorescent protein systems could monitor IFITM1 topology in real-time during viral entry, with complementary fragments placed at N- and C-terminal domains to confirm their relative orientation across the membrane . Hydrogen-deuterium exchange mass spectrometry combined with antibody epitope mapping could provide detailed structural information about which IFITM1 domains are exposed on each side of the membrane. CRISPR-mediated tagging of endogenous IFITM1 with minimal tags would allow monitoring of native protein dynamics without overexpression artifacts. For studying membrane integration kinetics, pulse-chase experiments using temporally controlled labeling systems coupled with domain-specific antibodies could track how newly synthesized IFITM1 acquires its final topology. Advanced biophysical techniques like solid-state NMR spectroscopy of isotopically-labeled IFITM1 in membrane mimetics could provide atomic-level structural information that antibody-based methods cannot resolve. Implementing these methodologies would resolve current contradictions in the literature regarding IFITM1 topology and its relationship to antiviral function.

How can researchers better integrate IFITM1 antibody data with functional genomics approaches?

Integrating IFITM1 antibody data with functional genomics requires systematic coordination between protein-level studies and genomic/transcriptomic analyses. Develop correlated workflows that couple CRISPR screening data with antibody-based validation, where hits from genome-wide screens for viral restriction factors are systematically validated using IFITM1-specific antibodies to confirm expression changes and localization patterns . Implement single-cell approaches that combine transcriptome analysis with immunofluorescence using IFITM1 antibodies to correlate mRNA expression heterogeneity with protein-level variation and functional outcomes. For regulatory element characterization, couple IFITM1 antibody-based chromatin immunoprecipitation (ChIP) of transcription factors with reporter assays to functionally validate predicted regulatory elements in the IFITM1 promoter region, particularly the ISRE and GAS elements . Create integrated datasets by performing quantitative proteomics on IFITM1 immunoprecipitates alongside RNA-seq analysis following viral infection to correlate changes in IFITM1 protein interactions with transcriptional responses. For structure-function analyses, combine deep mutational scanning of IFITM1 with high-throughput antibody-based phenotyping to comprehensively map functional domains and critical residues . When studying species-specific differences, use cross-species validated antibodies to compare IFITM1 expression patterns across evolutionarily diverse cell types alongside comparative genomics analysis to identify conserved functional elements.

How might single-molecule approaches using IFITM1 antibodies transform our understanding of its antiviral mechanisms?

Single-molecule approaches using IFITM1 antibodies offer transformative potential for understanding antiviral mechanisms at unprecedented resolution. Single-molecule tracking using quantum dot-conjugated Fab fragments derived from IFITM1 antibodies could reveal the dynamics of IFITM1 movement within membranes during viral challenge, potentially identifying restriction hotspots where IFITM1 molecules cluster to prevent viral fusion . Super-resolution techniques like PALM/STORM with specifically labeled antibodies could determine the stoichiometry of IFITM1 in functional antiviral complexes and quantify how this changes during infection. Single-molecule FRET experiments using dual-labeled antibodies could detect conformational changes in IFITM1 structure upon viral binding or during interferon stimulation, potentially revealing activation-dependent structural rearrangements . For studying molecular interactions, single-molecule pull-down assays using surface-immobilized IFITM1 antibodies followed by detection of fluorescently-labeled viral components could determine binding affinities and kinetics of these interactions. Correlative light and electron microscopy combining antibody-based fluorescence with nanoscale structural imaging could visualize the precise relationship between individual IFITM1 molecules and viral particles during entry restriction . Advanced techniques like single-molecule force spectroscopy using antibody-functionalized AFM tips could measure the mechanical properties of IFITM1-containing membranes, potentially revealing how these proteins alter membrane rigidity or curvature to prevent viral fusion.