HSP90AB1 Antibody

What is HSP90AB1 and why is it important in research?

HSP90AB1 (Heat Shock Protein 90 Alpha Family Class B Member 1) is a molecular chaperone protein that plays crucial roles in various cellular processes. It functions primarily in protein folding, maturation, activation, and degradation pathways. HSP90AB1 participates in regulating several signaling pathways involved in cell cycle progression, cell survival, and apoptosis. Its significance in research stems from its interactions with numerous client proteins, including many oncogenic proteins, making it a potential target for cancer therapy. Additionally, HSP90AB1 responds to environmental stressors such as heat shock and oxidative stress, with its expression being induced by various stress stimuli .

Research on HSP90AB1 antibodies is valuable for understanding these cellular processes and potential therapeutic applications. Unlike its paralog HSP90AA1, HSP90AB1 shows specific functions in certain biological contexts, such as viral infections, making it an important protein to distinguish and study independently.

What are the common applications for HSP90AB1 antibodies in research?

HSP90AB1 antibodies are utilized in multiple experimental approaches, each with specific protocols and optimization requirements:

These applications enable researchers to detect, quantify, and localize HSP90AB1 in various experimental systems . For research requiring specific detection of human HSP90AB1, recombinant monoclonal antibodies offer high specificity, as they typically detect only human HSP90AB1 protein without cross-reactivity to other species or related proteins.

How do HSP90AB1 and HSP90AA1 antibodies differ in their research applications?

Despite their structural similarities, HSP90AB1 and HSP90AA1 have distinct roles in cellular processes. These differences influence how their respective antibodies are used in research:

HSP90AB1 antibodies have shown particular relevance in viral infection studies, where HSP90AB1 knockdown significantly reduces infections like TGEV (Transmissible Gastroenteritis Virus), while HSP90AA1 knockdown shows no comparable effect . Additionally, HSP90AB1 has demonstrated specific interactions with immune signaling proteins like NFKB1, detectable through Proximity Ligation Assay using specific antibody pairs .

Recent studies show that HSP90α (encoded by HSP90AA1) is solely responsible for certain client proteins like hERG, suggesting that Hsp90α inhibition accounts more for cardio and ocular toxicities in clinical trials . This functional differentiation emphasizes the importance of using isoform-specific antibodies when studying particular cellular processes or developing targeted therapies.

When designing experiments, researchers should carefully select between these antibodies based on the specific pathway or process under investigation. Cross-validation with multiple antibodies targeting different epitopes may be necessary to confirm findings related to specific HSP90 isoforms.

How can I design effective knockdown experiments to study HSP90AB1 function?

Designing effective HSP90AB1 knockdown experiments requires careful consideration of several factors to ensure specific targeting and reliable interpretation of results:

For CRISPR-Cas9 based knockdown, sgRNA design is critical. Based on published research, effective sgRNA sequences targeting HSP90AB1 include:

• Forward: 5'-CACCGAGGTCAAAAGGAGCCCGACG-3'

• Reverse: 5'-AAACCGTCGGGCTCCTTTTGACCTC-3'

The knockdown efficiency should be validated using both qRT-PCR and Western blot, as transcript and protein reduction may not correlate perfectly. In published studies, HSP90AB1 knockdown has achieved approximately 55% reduction in mRNA levels and 20% reduction in protein levels . The discrepancy between mRNA and protein reduction highlights HSP90AB1's stability and potentially long half-life, requiring careful experimental timing for observing phenotypic effects.

Control experiments should include testing cell viability using methods like CCK-8 assay to ensure that observed phenotypes are not due to general cytotoxicity. Additionally, specific controls targeting related proteins (like HSP90AA1) help distinguish isoform-specific functions. This comparative approach has revealed that HSP90AB1, but not HSP90AA1, significantly impacts certain viral infections like TGEV .

What are the most effective protocols for detecting protein-protein interactions involving HSP90AB1?

Proximity Ligation Assay (PLA) represents one of the most sensitive methods for detecting HSP90AB1 protein interactions in situ. This technique visualizes protein-protein interactions as distinct fluorescent dots, each representing a single interaction complex.

For detecting HSP90AB1-NFKB1 interactions:

• Fix and permeabilize cells (HeLa cells work well for this application)

• Block non-specific binding sites

• Apply primary antibodies at optimized dilutions:

  • Anti-HSP90AB1 rabbit purified polyclonal antibody (1:1200)

  • Anti-NFKB1 mouse monoclonal antibody (1:50)

• Add PLA probes (secondary antibodies coupled to oligonucleotides)

• Add ligase and polymerase for rolling circle amplification

• Detect amplified signal using fluorescence microscopy

The resulting red fluorescent dots can be quantified using image analysis software like BlobFinder from Uppsala University . Critical controls include omitting one primary antibody, using non-interacting protein pairs, and validating interactions using complementary methods like co-immunoprecipitation.

For co-immunoprecipitation experiments, crosslinking before cell lysis can preserve transient interactions. Approximately 100-500 μg of total protein is typically required, with HSP90AB1 antibody concentrations of 2-5 μg per experiment generally providing optimal results for pulling down HSP90AB1 complexes.

How can HSP90AB1 antibodies be utilized to study its role in viral infections?

HSP90AB1 antibodies provide valuable tools for investigating the protein's involvement in viral lifecycle and host-virus interactions. Recent research has demonstrated HSP90AB1's critical role in certain viral infections, such as Transmissible Gastroenteritis Virus (TGEV).

To study HSP90AB1's role in viral infections, a comprehensive approach should include:

• Expression analysis: Track HSP90AB1 levels during infection using Western blot (1:500-1:2000 dilution) to determine if viral infection alters HSP90AB1 expression .

• Localization studies: Employ immunofluorescence (1:50-1:200 dilution) to visualize potential relocalization of HSP90AB1 during infection and assess colocalization with viral proteins .

• Functional studies: Combine HSP90AB1 knockdown (using verified sgRNAs) with viral infection assays. In TGEV studies, HSP90AB1 knockdown resulted in 42% reduction in viral gene expression, approximately 1 Log10 TCID50/mL reduction in viral titers, and 27% reduction in viral protein levels .

• Inhibitor studies: Compare HSP90AB1 knockdown effects with pharmacological inhibition using specific Hsp90 inhibitors like VER-82576 or KW-2478, providing complementary approaches to validate findings .

• Overexpression experiments: Express HSP90AB1 in cells to determine if increased levels enhance viral replication. Interestingly, while HSP90AB1 knockdown reduces TGEV infection, overexpression does not enhance viral replication, suggesting HSP90AB1 is necessary but not sufficient to boost infection .

These approaches provide a comprehensive framework for defining HSP90AB1's role in viral pathogenesis and may identify potential antiviral targets.

How can I resolve issues with non-specific binding when using HSP90AB1 antibodies?

Non-specific binding is a common challenge when working with HSP90AB1 antibodies, particularly due to the high homology between HSP90 family members. To improve antibody specificity and reduce background:

• Optimize blocking conditions: For Western blots, extending blocking time to 2 hours using 5% non-fat milk or 3% BSA can significantly reduce non-specific binding. For immunostaining applications, include 0.1-0.3% Triton X-100 in the blocking buffer to reduce cytoplasmic background.

• Antibody validation: Confirm antibody specificity using positive controls (cells known to express HSP90AB1) and negative controls (HSP90AB1 knockdown cells). Western blots should show a single band at approximately 83-84 kDa for HSP90AB1 .

• Cross-adsorption: If cross-reactivity with HSP90AA1 is observed, pre-adsorb the antibody with recombinant HSP90AA1 protein to remove antibodies that bind both isoforms. This is particularly important when studying specific isoform functions.

• Dilution optimization: Test a range of antibody dilutions beyond the recommended range. While manufacturer recommendations suggest 1:500-1:2000 for Western blot and 1:50-1:200 for IHC/IF/FC , optimal dilutions may vary depending on sample type and detection method.

• Incubation conditions: For Western blots, overnight incubation at 4°C often provides better signal-to-noise ratio than shorter incubations at room temperature.

If background persists despite these optimizations, consider using monoclonal antibodies which generally offer higher specificity than polyclonal antibodies, or switch to recombinant antibodies that have been specifically validated for human HSP90AB1 detection .

What controls should I include when studying HSP90AB1 in different experimental designs?

Proper controls are essential for generating reliable and interpretable data when studying HSP90AB1. Key controls should be tailored to specific experimental approaches:

For Western blot analysis:

• Positive control: Cell lines with confirmed HSP90AB1 expression (HeLa cells are commonly used)

• Loading control: Anti-ACTB (β-actin) antibody ensures equal protein loading

• Isoform control: Include HSP90AA1 detection to distinguish isoform-specific effects

For knockdown experiments:

• Non-targeting sgRNA/siRNA control: Accounts for non-specific effects of the knockdown procedure

• Related isoform knockdown (e.g., HSP90AA1): Distinguishes between isoform-specific functions

• Cell viability assessment: CCK-8 assay confirms that phenotypes aren't due to reduced cell viability

• Calculation: cell viability (%) = [(OD450 KD/OVER-OD450 blank)/(OD450 WT-OD450 blank)] × 100%

For overexpression studies:

• Empty vector control: Accounts for effects of transfection/transduction

• Verification of overexpression: qRT-PCR and Western blot to confirm increased expression levels

• Functional readouts: Appropriate assays to detect any gain-of-function effects

For proximity ligation assays:

• Single antibody controls: Omit one primary antibody to establish background signal levels

• Non-interacting protein pairs: Use antibodies against proteins not expected to interact with HSP90AB1

• Positive interaction control: Known HSP90AB1 interaction partner such as NFKB1

Including these controls enables more confident interpretation of results and helps distinguish between specific HSP90AB1-related effects and experimental artifacts or general cellular responses.

How should I interpret conflicting results from different detection methods for HSP90AB1?

Conflicting results across different detection methods for HSP90AB1 are not uncommon and require careful analysis to resolve. This systematic approach can help interpret such discrepancies:

• Compare protein vs. mRNA detection: HSP90AB1 knockdown experiments have shown disparities between mRNA reduction (55%) and protein reduction (20%) , indicating post-transcriptional regulation. When results conflict, protein-level measurements generally provide more functional relevance than transcript levels.

• Evaluate detection sensitivity thresholds: Different methods have varying sensitivity limits:

  • Western blot: Good for abundant proteins, typically detects >100,000 molecules/cell

  • qPCR: Highly sensitive for transcripts, can detect <100 copies

  • IHC/IF: Sensitivity depends on antibody affinity and signal amplification

  Low-abundance changes might be detected by more sensitive methods but missed by others.

• Consider spatial versus total expression: Immunostaining might show apparent upregulation in specific cellular compartments even when total protein levels measured by Western blot remain unchanged, revealing redistribution rather than expression changes.

• Examine temporal dynamics: Transient changes might be captured by one timepoint in one method but missed in another. For example, in viral infection studies, the optimal sampling timepoint is often 24 hours post-infection .

• Assess experimental conditions: Variables like cell confluence, passage number, and sample preparation can impact results. Standardize these conditions when comparing across methods.

When faced with conflicting results, the most robust approach is triangulation using multiple independent methods. For example, combining Western blot for protein levels, proximity ligation assay for protein interactions , and functional assays like viral infection studies can provide a more complete picture of HSP90AB1 biology.

How is HSP90AB1 involved in immune checkpoint blockade therapy resistance?

Recent research has uncovered a significant role for HSP90AB1 (Hsp90β) in immune checkpoint blockade (ICB) therapy resistance, opening new avenues for cancer immunotherapy. This relationship involves complex interactions between HSP90AB1, tumor cells, and the immune microenvironment:

Selective inhibition of HSP90AB1, unlike pan-HSP90 inhibition, appears to enhance interferon response and improve immunotherapy outcomes. Pan-HSP90 inhibitors have shown limited clinical success due to modest efficacy, dose-limiting toxicities (DLTs), and the induction of heat shock response (HSR) that counteracts the inhibitory effects . In contrast, selective HSP90AB1 inhibitors do not trigger HSR and demonstrate more favorable efficacy and safety profiles.

The mechanism appears to involve HSP90AB1's regulation of interferon signaling pathways. Inhibiting HSP90β upregulates interferon response genes, potentially converting "cold" tumors unresponsive to immunotherapy into "hot" immunogenic tumors. This conversion enhances T-cell infiltration and recognition of tumor cells, thereby improving response to immune checkpoint inhibitors .

Unlike pan-HSP90 inhibitors that target all four HSP90 isoforms and cause significant toxicities, HSP90β-selective inhibition appears safer. Notably, certain toxicities observed with pan-HSP90 inhibitors, particularly cardio and ocular toxicities, are attributed to HSP90α inhibition (encoded by HSP90AA1) rather than HSP90β inhibition . This isoform-specific effect highlights the potential benefit of selective targeting.

These findings suggest HSP90AB1-selective inhibition may represent a promising strategy to overcome ICB resistance in cancer patients who fail to respond to current immunotherapies.

What is the role of HSP90AB1 in viral pathogenesis and potential antiviral strategies?

HSP90AB1 plays a critical role in the lifecycle of various viruses, making it a promising target for broad-spectrum antiviral strategies. Recent research has elucidated several mechanisms through which HSP90AB1 facilitates viral infections:

In Transmissible Gastroenteritis Virus (TGEV) infections, HSP90AB1 has been identified as an essential host factor. Experimental knockdown of HSP90AB1 resulted in significant reduction of viral replication, with 42% lower viral M gene mRNA levels, approximately 1 Log10 TCID50/mL reduction in viral titers, and 27% decrease in viral N protein levels . Interestingly, while HSP90AB1 is necessary for efficient TGEV infection, its overexpression did not enhance viral replication, suggesting it functions as a required factor rather than a rate-limiting one .

This viral dependency appears to be isoform-specific. Unlike HSP90AB1, knockdown of the related HSP90AA1 had no significant effect on TGEV infection parameters . This specificity suggests that targeting HSP90AB1 may provide more selective antiviral effects with potentially fewer side effects compared to pan-HSP90 inhibition.

Pharmacological HSP90 inhibitors like KW-2478 and VER-82576 can be used experimentally to validate the role of HSP90AB1 in viral infections . The development of more selective HSP90AB1 inhibitors could provide new therapeutic options for viral diseases with reduced toxicity compared to pan-HSP90 inhibitors.

Future antiviral strategies may involve temporary HSP90AB1 inhibition during acute viral infections or designing inhibitors that specifically disrupt viral protein interactions with HSP90AB1 without affecting normal cellular functions.

How do protein-protein interactions involving HSP90AB1 contribute to disease pathogenesis?

HSP90AB1 engages in numerous protein-protein interactions (PPIs) that are increasingly recognized as critical in disease development. These interactions represent both valuable biomarkers and potential therapeutic targets:

A significant interaction partner of HSP90AB1 is NFKB1, a key transcription factor in inflammation and immune responses. This interaction can be visualized using Proximity Ligation Assay, which reveals distinct protein interaction complexes as fluorescent dots in cellular imaging . The HSP90AB1-NFKB1 interaction may influence inflammatory signaling pathways relevant to autoimmune disorders and certain cancers.

In cancer pathogenesis, HSP90AB1 stabilizes various oncogenic client proteins. Unlike its related isoform HSP90AA1, HSP90AB1 appears to have unique client specificities and functions. This specificity is particularly relevant for therapeutic development, as targeting HSP90AB1 may provide more precise intervention with fewer side effects than pan-HSP90 inhibition .

For immune checkpoint blockade therapy resistance, HSP90AB1 interactions with components of interferon signaling pathways appear crucial. Disrupting these interactions through selective HSP90AB1 inhibition can upregulate interferon response genes and potentially enhance immunotherapy efficacy .

Understanding the structural basis of these interactions is advancing through techniques like hydrogen-deuterium exchange mass spectrometry, which can map interaction interfaces. This knowledge facilitates the design of selective inhibitors that disrupt specific pathological interactions while preserving normal HSP90AB1 functions.

Future therapeutic approaches may involve targeting specific HSP90AB1 protein-protein interactions rather than inhibiting the chaperone activity entirely, potentially offering greater selectivity and reduced toxicity compared to current strategies.

How should I analyze HSP90AB1 expression data across different tissue or cell types?

Analyzing HSP90AB1 expression across diverse tissue or cell types requires a structured approach to account for biological variables and technical considerations:

Start by establishing appropriate normalization strategies. When comparing HSP90AB1 expression:

• For qPCR: Normalize to multiple housekeeping genes stable across your sample types, not just a single reference gene

• For Western blot: Normalize to loading controls like β-actin (ACTB) , but be aware that housekeeping protein expression can vary between tissues

• For immunostaining: Use tissue-specific positive controls and standardize image acquisition parameters

Consider tissue-specific baseline expression levels using reference databases. HSP90AB1 expression varies naturally between tissues, with database identifiers such as:

• UniGene: Hs.509736

• HGNC: 5258

• OMIM: 140572

• KEGG: hsa:3326

• STRING: 9606.ENSP00000325875

These resources provide reference expression patterns across normal tissues.

For differential expression analysis, use appropriate statistical tests based on your experimental design:

• Paired samples: Paired t-test or Wilcoxon signed-rank test

• Multiple groups: ANOVA with post-hoc tests or Kruskal-Wallis for non-parametric data

• Correlation with functional outcomes: Consider regression analysis

When interpreting results, consider that HSP90AB1 function may differ between tissues even at similar expression levels due to tissue-specific client proteins or co-chaperones. Additionally, changes in subcellular localization (detectable by immunofluorescence) may represent functional changes without altered total expression.

Finally, validate key findings using orthogonal methods. If Western blot shows differential expression, confirm with qPCR and functional assays to establish biological significance beyond statistical differences.

What bioinformatic approaches are recommended for analyzing HSP90AB1-related interaction networks?

Bioinformatic analysis of HSP90AB1 interaction networks requires sophisticated approaches to capture the complexity of its chaperone functions and client interactions:

Start with database mining using HSP90AB1's established identifiers (HGNC: 5258, STRING: 9606.ENSP00000325875) . STRING database analysis provides a foundation for network construction, offering evidence-based protein-protein interactions with confidence scores. Focus on high-confidence interactions (score >0.7) to reduce false positives.

For network visualization and analysis, tools like Cytoscape combined with plug-ins such as MCODE or ClusterONE help identify functional modules within the larger HSP90AB1 interactome. Color-code nodes based on:

• Cellular compartment

• Biological process

• Client vs. co-chaperone status

• Disease association

Perform pathway enrichment analysis using tools like KEGG, Reactome, or Gene Ontology to identify biological processes overrepresented in the HSP90AB1 network. Compare enrichment patterns between different experimental conditions to identify condition-specific shifts in HSP90AB1 function.

For experimental validation of predicted interactions, prioritize candidates using:

• Network centrality measures (identifying hub proteins)

• Differential expression in your experimental system

• Disease relevance based on literature

• Druggability for potential therapeutic targeting

Validate top candidates using proximity ligation assay, which has successfully demonstrated HSP90AB1-NFKB1 interactions .

For more advanced analysis, consider integrating multiple omics data types:

• Combine proteomic data on HSP90AB1 interactors with transcriptomic data

• Include phosphoproteomic data to identify post-translational regulation

• Incorporate structural data to predict interaction interfaces

Machine learning approaches can also predict novel HSP90AB1 interactors based on features of known clients, potentially identifying interactions missed by experimental approaches.

How do I reconcile contradictory findings about HSP90AB1 function in different disease models?

Contradictory findings regarding HSP90AB1 function across disease models are common in the literature and require careful analysis to reconcile. This analytical framework helps researchers navigate these discrepancies:

First, evaluate context-dependent factors that might explain divergent results:

• Cell/tissue type differences: HSP90AB1 functions are often tissue-specific due to varying client protein expression. For example, HSP90AB1 has specific effects in viral infections that differ from its roles in cancer cells .

• Disease stage: HSP90AB1's role may evolve during disease progression. Early cytoprotective effects might become pathological in advanced disease states.

• Experimental methodology: Knockdown approaches (55% mRNA reduction, 20% protein reduction) versus pharmacological inhibition may affect different HSP90AB1 functions.

• Isoform specificity: Ensure studies distinguished between HSP90AB1 and HSP90AA1, as they have different client specificities despite structural similarity .

In cancer research, contradictory findings are particularly common. HSP90AB1 inhibition reduces tumor growth in some models while potentially enhancing immune responses in others . These seemingly contradictory effects may actually represent complementary mechanisms, with direct anti-tumor effects combining with enhanced immune recognition.

In viral infection studies, HSP90AB1 knockdown significantly reduced TGEV replication, yet overexpression did not enhance viral replication . This apparent contradiction suggests HSP90AB1 functions as a necessary but not sufficient factor for viral replication, highlighting the complexity of its role.

When integrating conflicting literature, develop testable models that might explain discrepancies, such as:

• Dose-dependent biphasic effects

• Cell type-specific co-factor availability

• Temporal dynamics of HSP90AB1 function during disease progression

• Different HSP90AB1 domains mediating distinct functions

Advanced experimental approaches, such as domain-specific mutations or time-resolved studies, can test these hypotheses and potentially reconcile contradictory findings into a more unified model of HSP90AB1 function.