HEATR9 Antibody

What is HEATR9 and why is it significant in viral infection research?

HEATR9 (Heat Repeat-Containing Protein 9) is a novel gene identified as a virus and cytokine-inducible viral responsive gene. Its significance in viral infection research stems from its dramatic upregulation during respiratory viral infections, particularly with influenza virus and respiratory syncytial virus (RSV) . HEATR9 is nearly undetectable in uninfected cells (qRT-PCR Ct values >35 cycles) but can be upregulated more than 1000-fold following influenza virus infection .

The protein contains HEAT repeats, which are conserved tertiary structures consisting of two amphiphilic helices connected by an intra-unit loop. While the presence of these domains doesn't directly indicate function, HEAT repeats are found in proteins associated with DNA structure and transcriptional control, suggesting HEATR9 may regulate gene expression during infection .

Most importantly, HEATR9 has been shown to affect chemokine expression, with knockdown experiments demonstrating that it plays a role in cytokine production, specifically regulating chemokine ligands that control immune cell recruitment and inflammation . This makes HEATR9 a potentially important target for understanding host-pathogen interactions and the inflammatory response to viral infections.

What applications are most suitable for HEATR9 antibodies in respiratory virus research?

HEATR9 antibodies are particularly valuable for several research applications involving respiratory viruses:

• Expression Studies: Western blot analysis to detect and quantify HEATR9 protein levels in infected versus uninfected cells . This is especially useful given HEATR9's dramatic upregulation during infection.

• Infection Monitoring: HEATR9 antibodies can be used to monitor the progression of viral infection in both in vitro and in vivo models, as HEATR9 expression correlates with viral burden .

• Host Response Characterization: Immunostaining to visualize the cellular localization of HEATR9 during various stages of infection, helping to determine its role in the host response .

• Cytokine Regulation Studies: Investigating HEATR9's role in regulating specific chemokines, particularly CCL4 and CCL5, during viral infection .

• Bystander Cell Effects: Examining how HEATR9 is upregulated in both directly infected cells and bystander cells, which can provide insights into infection signaling mechanisms .

When designing experiments with HEATR9 antibodies, researchers should consider the timing of sample collection, as HEATR9 expression peaks at specific timepoints during infection (e.g., day 6 for influenza infection in mouse models) .

What sample preparation methods yield optimal results when using HEATR9 antibodies?

For optimal results with HEATR9 antibodies, the following sample preparation methods are recommended:

For Western Blot Analysis:

• Properly isolate proteins from cell lysates using a buffer containing protease inhibitors to prevent degradation

• Recommended dilution range for HEATR9 antibodies is typically 1:500-2000 for Western blot applications

• For human/mouse samples, using antibodies targeting the 505-555 amino acid region has shown good specificity

For Tissue Samples:

• For RNA isolation (when correlating protein with transcript levels), tissues should be collected and processed immediately to preserve RNA integrity

• Flash freezing of tissues followed by homogenization is effective for both protein and RNA extraction

• When working with lung tissue, proper dissection to isolate alveolar epithelial cells, where HEATR9 is highly expressed, is crucial

For Infected Cell Culture:

• When infecting A549 cells (commonly used for HEATR9 studies), a multiplicity of infection (MOI) of 1 for influenza virus or RSV has been shown to effectively induce HEATR9 expression

• Collecting samples at multiple timepoints is advised, as HEATR9 expression changes dynamically during infection

How do HEATR9 expression patterns differ across tissue types and infection states?

HEATR9 exhibits distinct expression patterns across different tissues and infection states:

Cellular Distribution in Lungs:
HEATR9 is particularly upregulated in the lung during respiratory infections, with significant expression in alveolar epithelial cells (AECs) . In influenza virus infection models, two distinct AEC populations show different levels of HEATR9 upregulation:

Temporal Expression Pattern:
In influenza-infected mouse lungs, HEATR9 expression follows a specific pattern:

In vitro vs. In vivo Expression:
While direct viral infection provides the strongest HEATR9 induction, the protein can also be upregulated by treatment with:

• Infected cell culture supernatants (suggesting cytokine-mediated induction)

• Combinations of inflammatory cytokines, though to a lesser extent than direct infection

These patterns suggest HEATR9 responds both to direct viral sensing mechanisms and to secondary inflammatory signals, making it an important marker for both direct and indirect effects of viral infections.

What are the critical considerations for validating HEATR9 antibody specificity?

Validating the specificity of HEATR9 antibodies is crucial for ensuring reliable research outcomes. Several critical considerations should be addressed:

Epitope Selection and Antibody Design:

• The immunogen selection is critical - antibodies targeting the C-terminal region (505-555 amino acids) of HEATR9 have shown good specificity

• For polyclonal antibodies, the synthetic peptide sequence "KLKNKVLSVYEAPKTNVKAEPTRFQKEPENPEELTIQDFRLAKLNPLFIA" has been effectively used as an immunogen

Validation Controls:

• Positive Controls: Using 293T whole cell lysates as positive controls for Western blot applications

• Knockdown Validation: Employing HEATR9 knockdown samples to confirm antibody specificity

• Cross-reactivity Testing: When using antibodies across species (human/mouse), cross-reactivity should be thoroughly validated

Conformational Epitope Considerations:
When developing assays to detect HEATR9, it's important to ensure that key conformational epitopes are maintained. For instance, in ELISA development, antigen coating conditions should preserve important conformational epitopes . This can be validated using inhibition ELISAs with reference monoclonal antibodies that recognize specific epitopes.

Technical Validation Methods:

• Multiple antibodies targeting different regions of HEATR9 should ideally be compared

• Combining immunodetection with transcript analysis (qRT-PCR) to correlate protein and mRNA levels

• When using the antibody in novel applications beyond those specified by manufacturers (typically Western blot), additional validation steps should be performed

Researchers should be aware that there are multiple splice variants of HEATR9 in humans (though mice have only one transcript) , which may affect antibody binding and detection, requiring careful consideration when interpreting results.

How can HEATR9 antibodies be used to investigate the protein's role in chemokine regulation during viral infection?

HEATR9 antibodies can be employed in several sophisticated experimental approaches to elucidate its role in chemokine regulation during viral infections:

Immunoprecipitation-Based Approaches:

• Co-immunoprecipitation (Co-IP) using HEATR9 antibodies can identify protein interaction partners involved in chemokine expression pathways

• Chromatin immunoprecipitation (ChIP) assays can determine if HEATR9 directly interacts with promoter regions of chemokine genes, given that HEAT repeats are associated with transcriptional control

Combined Knockdown and Antibody Detection Studies:
Research has shown that HEATR9 knockdown affects chemokine expression, particularly CCL4 and CCL5 . A comprehensive experimental approach would involve:

• siRNA or shRNA-mediated knockdown of HEATR9

• Infection with influenza virus or RSV

• Use of HEATR9 antibodies to confirm knockdown efficiency

• Quantification of chemokine expression using qRT-PCR and ELISA

• Correlation analysis between HEATR9 protein levels and chemokine production

Pathway Analysis:

• Phosphorylation-specific antibodies can be used alongside HEATR9 antibodies to determine if HEATR9 influences signaling pathways known to regulate chemokine expression

• Immunofluorescence co-localization studies can reveal if HEATR9 associates with transcription factors involved in chemokine gene expression

Temporal Dynamics:
Using HEATR9 antibodies for time-course experiments can reveal how HEATR9 protein levels correlate with the kinetics of chemokine expression. Evidence suggests that HEATR9 may act as a checkpoint for chemokine expression, as knockdown reduces specific chemokine genes .

SNP-Associated Studies:
Research has identified single nucleotide polymorphisms (SNPs) associated with both CCL5 and HEATR9 expression . Using HEATR9 antibodies to quantify protein levels in cells with different SNP variants could provide insights into how genetic variations affect HEATR9's role in chemokine regulation.

What approaches can be used to study HEATR9 protein-protein interactions and potential binding partners?

Studying HEATR9 protein-protein interactions requires specialized approaches due to its infection-inducible nature and potential regulatory functions. Here are methodological approaches:

Affinity Purification-Mass Spectrometry (AP-MS):

• Immunoprecipitation using HEATR9 antibodies followed by mass spectrometry can identify binding partners

• This approach should be performed under both uninfected and infected conditions to identify infection-specific interactions

• Crosslinking prior to immunoprecipitation may be necessary to capture transient interactions

Proximity-Based Labeling Approaches:

• BioID or APEX2 fusion proteins with HEATR9 can identify proteins in close proximity under physiological conditions

• These approaches are particularly valuable for studying HEATR9 interactions in the context of transcriptional complexes, as suggested by its HEAT repeat domains

Split-Reporter Assays:

• Bimolecular Fluorescence Complementation (BiFC) or split-luciferase assays with HEATR9 and candidate interactors

• This approach can visualize where in the cell these interactions occur, which is important given HEATR9's potential role in transcriptional regulation

Domain-Specific Interaction Mapping:
Since HEATR9 contains HEAT repeat domains, which are known to mediate protein-protein interactions, a domain-specific approach is warranted:

Functional Validation:

• Co-knockdown experiments of HEATR9 and identified interactors

• Rescue experiments with mutated versions of HEATR9 lacking specific interaction domains

• CRISPR/Cas9-mediated tagging of endogenous HEATR9 for physiological interaction studies

When using antibodies for interaction studies, it's crucial to validate that the antibody epitope is not within an interaction interface, which could block binding and lead to false negatives.

How does HEATR9 expression correlate with anti-hemagglutinin stalk antibody responses during influenza infection?

The relationship between HEATR9 expression and anti-hemagglutinin (HA) stalk antibody responses represents an intriguing area at the intersection of innate and adaptive immunity during influenza infection.

Correlation Analysis During Infection:
Research on anti-HA stalk antibodies has shown that these antibodies are present before challenge and rise in response to influenza virus infection in approximately 64% of individuals . This pattern mirrors the induction of HEATR9, which is also dramatically upregulated during influenza infection . While direct correlative studies between HEATR9 and anti-HA stalk antibodies haven't been specifically documented, their parallel induction suggests potential mechanistic relationships.

Protection Correlates:
Anti-HA stalk antibody titers have been found to correlate with protection against certain aspects of influenza disease:

Methodological Approach for Investigation:
To investigate potential relationships between HEATR9 and anti-HA stalk antibodies, researchers could:

• Perform time-course analysis measuring both HEATR9 expression (using antibodies against HEATR9) and anti-HA stalk antibody titers following influenza infection

• Compare HEATR9 expression levels in responders versus non-responders to anti-HA stalk antibody production

• Examine whether HEATR9 knockdown affects the production of anti-HA stalk antibodies

• Investigate if individuals with certain HEATR9 SNPs show differential anti-HA stalk antibody responses

Considerations for Experimental Design:
When designing such studies, it's important to note that approximately 10-30% of healthy individuals have poor hemagglutination inhibition (HAI) responses after vaccination, with even worse response rates in the elderly . This could potentially correlate with HEATR9 expression patterns or polymorphisms, representing an important area for future research.

What methodological approaches can integrate CRISPR/Cas9 genome editing with HEATR9 antibody applications for functional studies?

Combining CRISPR/Cas9 genome editing with HEATR9 antibody applications offers powerful approaches for functional characterization of this infection-responsive gene. Here are methodological approaches:

Endogenous Tagging of HEATR9:
Using CRISPR/Cas9 to insert epitope tags (FLAG, HA, etc.) at the C-terminal end of the endogenous HEATR9 gene allows for:

• Detection using well-characterized commercial tag antibodies

• Immunoprecipitation studies under physiological expression conditions

• Live-cell imaging when using fluorescent protein tags

This approach has been successfully demonstrated for antibody development, as described in search result : "Using CRISPR/Cas9 genomic editing, we developed a simple and novel approach to produce site-specifically modified antibodies. A sortase tag was genetically incorporated into the C-terminal end of the third immunoglobulin heavy chain constant region (CH3) within a hybridoma cell line..." .

Generation of HEATR9 Knockout and Knockin Cell Lines:
CRISPR/Cas9 can be used to generate:

• Complete HEATR9 knockout cell lines for loss-of-function studies

• Cell lines expressing HEATR9 variants with specific domain deletions or mutations

• Knockin lines with inducible HEATR9 expression

These engineered cell lines can then be validated and characterized using HEATR9 antibodies to confirm:

• Complete absence of protein in knockout lines

• Expression levels in knockin variants

• Subcellular localization of mutant proteins

Mechanistic Studies Using Combined Approaches:

ChIP-Seq Integration:
For investigating HEATR9's potential role in transcriptional regulation:

• Use CRISPR/Cas9 to insert a tag suitable for ChIP (e.g., HA or FLAG)

• Perform ChIP-seq using antibodies against the tag

• Identify genomic binding sites, particularly at chemokine gene loci

• Validate findings using independent HEATR9 antibodies

Considerations for Experimental Design:
When using CRISPR/Cas9-modified systems alongside antibody applications, researchers should:

• Always include proper controls (unedited cells, mock-edited cells)

• Validate genome editing using both DNA sequencing and protein detection with antibodies

• Consider potential off-target effects of CRISPR/Cas9 editing

• Account for clonal variations by examining multiple successfully edited clones

What are the key technical challenges when using HEATR9 antibodies in multiplexed immunoassays?

Implementing HEATR9 antibodies in multiplexed immunoassays presents several technical challenges that require careful consideration:

Cross-Reactivity Concerns:

• When combining multiple antibodies in a single assay, cross-reactivity between antibodies must be thoroughly assessed

• HEATR9 antibodies should be tested against other targets in the multiplex panel, particularly other HEAT repeat-containing proteins

• Isotype compatibility must be considered to ensure secondary antibodies don't cross-react

Dynamic Range Limitations:
HEATR9 expression varies dramatically between uninfected and infected states (>1000-fold increase) , creating challenges for multiplex detection:

• Ensuring the assay can accurately quantify both very low (baseline) and very high (infection-induced) levels

• Balancing signal strength across all targets in the panel when HEATR9 may be overexpressed compared to other markers

• Implementing appropriate dilution series for samples with varying HEATR9 expression levels

Antibody Pairing Optimization:
For sandwich-based multiplex assays:

• Identifying optimal capture and detection antibody pairs for HEATR9

• Ensuring epitopes don't overlap with other antibodies in the panel

• Validating that conformational epitopes are maintained in the multiplex format

Sample Preparation Harmonization:
Different targets in a multiplex panel may require different sample preparation methods:

• HEATR9 detection may require specific lysis buffers to effectively extract the protein

• Ensuring sample preparation methods don't interfere with detection of other targets

• Standardizing fixation protocols that preserve HEATR9 epitopes while maintaining compatibility with other targets

Analytical Validation Strategy:
A comprehensive validation approach should include:

• Spike-recovery experiments with recombinant HEATR9 protein

• Calibration curves in both simple and complex matrices

• Comparison of HEATR9 measurements in single-plex versus multiplex formats

• Limit of detection determination specifically for HEATR9 in the multiplex context

Technical Solution Approaches:

• Using species-specific secondary antibodies when combining antibodies from different host species

• Implementing blocking steps with irrelevant immunoglobulins to reduce non-specific binding

• Considering sequential detection approaches for targets with vastly different expression levels

• Employing unique labels (fluorophores, enzymes) with minimal spectral overlap

How can researchers optimize HEATR9 antibody-based assays for quantifying infection-induced expression changes?

Optimizing HEATR9 antibody-based assays for quantifying infection-induced expression changes requires specialized approaches due to the dramatic upregulation during infection:

Dynamic Range Optimization:

• Implement wide dynamic range detection systems capable of accurately measuring >1000-fold changes

• Use log-scale calibration curves rather than linear scales

• Consider dilution series approaches for infected samples to ensure measurements fall within the linear range of detection

Temporal Considerations:
Research has shown that HEATR9 expression follows specific kinetics during infection . Optimization should include:

• Multiple time-point sampling to capture peak expression

• Correlation with viral burden measurements

• Standardization of collection times post-infection

Reference Standards Development:

• Generate recombinant HEATR9 protein standards for absolute quantification

• Develop standardized positive controls from infected cell lysates with known HEATR9 expression levels

• Consider synthetic peptide standards for mass spectrometry-based approaches

Normalization Strategies:
When quantifying HEATR9 expression changes, appropriate normalization is critical:

Assay-Specific Optimizations:

For Western Blot:

• Determine linear detection range for HEATR9 using recombinant protein standards

• Optimize protein loading amounts (lower for infected samples)

• Consider gradient gels to better resolve HEATR9 protein

• Implement quantitative Western blot approaches with infrared or chemiluminescent detection systems

For ELISA/Immunoassays:

• Develop sandwich ELISA using antibodies targeting different HEATR9 epitopes

• Optimize coating concentration and blocking conditions to maximize sensitivity

• Determine appropriate sample dilution factors for infected versus uninfected samples

• Validate with recombinant HEATR9 protein spike-in experiments

For Flow Cytometry:

• Optimize fixation and permeabilization protocols for intracellular HEATR9 staining

• Implement appropriate compensation controls when multiplexing with viral markers

• Consider cell sorting of infected versus uninfected populations for comparative analysis

What controls and validation experiments are essential when using HEATR9 antibodies in viral infection models?

When using HEATR9 antibodies in viral infection models, comprehensive controls and validation experiments are essential to ensure reliable and interpretable results:

Antibody Validation Controls:

• Specificity Controls:

  • HEATR9 knockout or knockdown cells/tissues as negative controls

  • Pre-absorption of antibody with immunizing peptide to confirm specificity

  • Multiple antibodies targeting different HEATR9 epitopes to confirm findings

• Technical Controls:

  • Secondary antibody-only controls to assess non-specific binding

  • Isotype controls matched to the HEATR9 antibody host and isotype

  • Titration experiments to determine optimal antibody concentration

Infection Model Controls:

• Viral Infection Validation:

  • Confirmation of successful infection using established viral markers

  • Appropriate mock-infected controls processed identically to infected samples

  • Positive controls using well-characterized viral response genes (e.g., ISG15)

• Time-Course Controls:

  • Multiple time-points to capture HEATR9 expression dynamics

  • Correlation with viral burden measurements

  • Inclusion of both early and late infection time-points

Experimental Design Validation:

• Sample Processing Validation:

  • Comparison of different lysis/extraction methods to ensure complete HEATR9 recovery

  • RNA and protein extraction from the same samples to correlate transcript and protein levels

  • Assessment of sample stability under storage conditions

• Quantification Method Validation:

  • Standard curves using recombinant HEATR9 protein

  • Technical replicates to assess assay reproducibility

  • Comparison of different detection methods (e.g., chemiluminescence vs. fluorescence)

Biological Validation Experiments:

• Functional Validation:

  • Correlation of HEATR9 protein levels with chemokine expression

  • Genetic manipulation (overexpression/knockdown) to confirm antibody detection specificity

  • Assessment of HEATR9 in both directly infected and bystander cells

• Cross-Species Validation:

  • When using antibodies across species (human/mouse), validation in each species

  • Species-specific positive controls

  • Comparison with species-specific qRT-PCR data

Critical Additional Controls for Specific Applications:

Proper implementation of these controls ensures that findings related to HEATR9 in viral infection models are robust, reproducible, and accurately represent the biological phenomena being studied.

What emerging technologies could enhance the study of HEATR9 beyond traditional antibody applications?

Several cutting-edge technologies show promise for advancing HEATR9 research beyond conventional antibody-based approaches:

CRISPR-Based Technologies:

• CRISPR Activation/Interference Systems: CRISPRa/CRISPRi for modulating HEATR9 expression without genetic modification

• CRISPR Screening: Genome-wide screens to identify regulators and interaction partners of HEATR9

• Base Editing: Precise modification of HEATR9 SNPs associated with chemokine regulation

• Prime Editing: Introduction of specific mutations to study structure-function relationships in HEATR9

Proximity Labeling Technologies:

• TurboID/miniTurboID: Rapid biotin labeling of proteins proximal to HEATR9 under various infection conditions

• APEX2: Subcellular-specific mapping of HEATR9 interactome during different infection phases

• Split-TurboID: Detection of specific HEATR9 protein-protein interactions in living cells

Advanced Imaging Approaches:

• Super-Resolution Microscopy: Nanoscale visualization of HEATR9 localization during infection

• Lattice Light-Sheet Microscopy: Live-cell imaging of HEATR9 dynamics during viral entry and replication

• Correlative Light and Electron Microscopy (CLEM): Combining ultrastructural context with HEATR9-specific labeling

Single-Cell Technologies:

• Single-Cell Proteomics: Quantifying HEATR9 protein levels in heterogeneous infected cell populations

• Single-Cell Transcriptomics with Protein Detection: Simultaneous measurement of HEATR9 mRNA and protein

• Spatial Transcriptomics: Mapping HEATR9 expression patterns in tissue contexts during infection

Protein Structure and Interaction Technologies:

• AlphaFold2/RoseTTAFold: Computational prediction of HEATR9 structure and interaction surfaces

• Hydrogen-Deuterium Exchange Mass Spectrometry: Mapping HEATR9 protein dynamics and conformational changes

• Cryo-EM: Structural determination of HEATR9 complexes with interaction partners

Methodological Integration Approaches:

Emerging Considerations:
Recent developments in computational antibody design, as mentioned in search result , could enable the development of highly specific engineered antibodies against HEATR9 with tailored properties. Approaches like GaluxDesign, RFantibody, and dyMEAN offer promising avenues for generating binders with improved specificity and functionality .

The integration of these technologies will enable more comprehensive understanding of HEATR9's role in viral infection and potentially uncover novel functions beyond its currently known involvement in chemokine regulation.

How might HEATR9 research contribute to understanding broader host-pathogen interaction networks?

HEATR9 research has significant potential to advance our understanding of host-pathogen interaction networks through several key pathways:

Integration into Viral Sensing Pathways:
HEATR9 is induced by both direct viral infection and cytokine stimulation , positioning it at the intersection of multiple viral sensing pathways. Investigating how HEATR9 fits into these networks could reveal:

• New connections between pattern recognition receptor signaling and chemokine regulation

• Previously unrecognized signaling nodes in the host antiviral response

• Integration points between type I interferon responses and chemokine-mediated inflammation

Chemokine Regulatory Networks:
HEATR9 knockdown affects chemokine expression, particularly CCL4 and CCL5 , suggesting it may function as a regulatory node in inflammation networks:

• Mapping HEATR9's position in chemokine regulatory cascades could identify novel therapeutic targets

• Understanding how HEATR9 influences the balance between protective immunity and immunopathology

• Determining whether HEATR9 serves as a checkpoint for specific subsets of inflammatory mediators

Transcriptional Regulation Complexes:
The presence of HEAT repeat domains suggests potential involvement in transcriptional regulation :

• HEATR9 may be part of transcriptional complexes controlling infection-responsive gene expression

• Investigating HEATR9's potential role in chromatin remodeling during infection

• Determining if HEATR9 interacts with specific transcription factors involved in antiviral responses

Bystander Cell Communication Networks:
HEATR9 is upregulated in both directly infected cells (481-fold) and bystander cells (203-fold) , suggesting involvement in infection-associated cell-to-cell communication:

• HEATR9 may help coordinate responses between infected and uninfected neighboring cells

• It could be part of alarm signaling networks that prepare surrounding cells for infection

• Understanding how HEATR9 contributes to tissue-level antiviral defense coordination

Systems Biology Integration:
Combining HEATR9 research with systems-level analyses could yield significant insights:

Genetic Variation and Disease Susceptibility:
Research has identified SNPs associated with both CCL5 and HEATR9 expression , suggesting genetic links to disease outcomes:

• Investigating how HEATR9 genetic variations influence susceptibility to various infectious diseases

• Understanding if HEATR9 polymorphisms affect the severity of respiratory infections

• Determining whether HEATR9 genetic variants could predict vaccine responsiveness

By advancing our understanding of HEATR9 in these contexts, researchers can build more comprehensive models of host-pathogen interactions, potentially identifying new therapeutic targets and improving predictions of disease outcomes based on host response patterns.