HSPA4 Monoclonal Antibody

What is HSPA4 and why is it significant in research?

HSPA4 is an 840 amino acid chaperone protein, also known as epididymis secretory sperm binding protein Li 5a and APG-2. It plays a dual role in cellular protein homeostasis by protecting existing proteins against aggregation and mediating correct folding of newly synthesized proteins . The significance of HSPA4 in research has grown substantially as evidence mounts for its involvement in various pathological conditions, particularly in cancer. While HSPA4 has a predicted molecular weight of approximately 94 kDa, it typically appears as a band of approximately 110 kDa in western blotting, likely due to post-translational modifications . Recent studies have established its potential role as a biomarker, especially in cutaneous malignant melanoma (CMM), where its expression is significantly upregulated compared to normal skin tissues .

What are the primary applications of HSPA4 monoclonal antibodies in basic research?

HSPA4 monoclonal antibodies are versatile tools in basic research with several key applications. Western blotting represents the most validated application, with recommended dilutions typically ranging from 1/50 to 1/800 depending on the specific antibody clone and experimental conditions . In this context, HSPA4 antibodies can detect a characteristic band of approximately 110 kDa in various cell lysates, such as MCF7 cells under reducing conditions . Beyond western blotting, these antibodies have been instrumental in immunohistochemistry studies to compare HSPA4 protein expression between normal and malignant tissues. For instance, in the Human Protein Atlas database, HSPA4 antibody staining revealed that 10 out of 12 CMM samples showed positive staining (ranging from weak to strong), while normal melanocytes consistently showed negative staining .

How should researchers design validation experiments for HSPA4 monoclonal antibodies?

Validation of HSPA4 monoclonal antibodies requires a multi-faceted approach to ensure specificity and reliability. Researchers should begin with western blotting validation using positive control cell lines known to express HSPA4, such as MCF7 cells . This should be complemented with negative controls, either through HSPA4 knockdown experiments or using cell lines with minimal HSPA4 expression. Validation experiments should assess:

• Specificity: Confirm the antibody detects a band of the expected size (~110 kDa for HSPA4) without significant non-specific binding

• Sensitivity: Determine the minimum amount of protein required for detection

• Reproducibility: Ensure consistent results across multiple experiments

• Cross-reactivity: Test the antibody against closely related heat shock proteins

Including siRNA knockdown of HSPA4 as a negative control is particularly valuable, as demonstrated in studies where HSPA4 knockdown in A-375 melanoma cells not only validated antibody specificity but also revealed functional consequences like reduced cell proliferation .

How does HSPA4 expression correlate with tumor progression and patient outcomes?

These findings were validated in independent datasets (GSE65094), confirming that elevated HSPA4 expression is a consistent predictor of poor prognosis. Receiver operating characteristic (ROC) curve analysis further demonstrated HSPA4's diagnostic value in distinguishing tumor tissue from normal tissue with good accuracy and sensitivity (AUC = 0.812) . This suggests HSPA4 has potential utility as both a diagnostic and prognostic biomarker in CMM.

What mechanisms underlie HSPA4's role in cancer progression?

The mechanisms through which HSPA4 contributes to cancer progression are multifaceted and involve both direct effects on tumor cells and modulation of the tumor microenvironment. Functional studies using siRNA-mediated knockdown of HSPA4 in melanoma cell lines have demonstrated that reducing HSPA4 expression significantly inhibits tumor cell proliferation, with effects becoming particularly pronounced four days after knockdown (P < 0.05) . This indicates that HSPA4 plays a direct role in promoting cancer cell growth.

Beyond its direct effects on tumor cells, HSPA4 appears to influence the tumor immune microenvironment in ways that may facilitate immune evasion. Analysis of the relationship between HSPA4 expression and immune cell infiltration revealed that HSPA4 expression is negatively correlated with most immune cell populations, including B cells, NK cells, dendritic cells, and cytotoxic T cells . Interestingly, it shows positive correlation with T helper cells, central memory T cells (Tcm), and Th2 cells . Tumors with high HSPA4 expression exhibited significantly lower stromal scores, immune scores, and ESTIMATE scores compared to those with low HSPA4 expression (P < 0.001) . These findings suggest that HSPA4 may promote an immunosuppressive microenvironment that facilitates tumor progression.

Gene Set Enrichment Analysis (GSEA) further indicated that HSPA4 and its related proteins are involved in the negative regulation of multiple immune-related signaling pathways, including those associated with interleukins (IL-2, IL-4, IL-12, IL-17, IL-18, IL-23, IL-27), T-cell receptor signaling, and immune checkpoint pathways like PD-1 . This broad immunomodulatory profile suggests that HSPA4 may contribute to immune suppression and immune escape mechanisms in the tumor microenvironment.

How should researchers interpret protein interaction networks involving HSPA4?

Protein interaction network analysis reveals that HSPA4 functions within a complex web of molecular interactions, particularly with other heat shock proteins and co-chaperones. Researchers should interpret these networks by focusing on the functional significance of key interactions rather than mere association data. Bioinformatic analyses have identified that HSPA4 closely interacts with DnaJ heat shock protein family members, particularly DNAJB1 and DNAJB6 .

When interpreting these networks, researchers should consider:

• Co-expression patterns: The expression of DNAJB1 is positively correlated with HSPA4 expression, suggesting coordinated functional roles

• Pathway context: HSPA4 interactions should be interpreted within the context of specific signaling pathways, such as protein folding, stress response, and immune modulation

• Disease relevance: Focus on interactions that show altered patterns in disease states compared to normal tissues

• Experimental validation: Network predictions should be validated through co-immunoprecipitation or proximity ligation assays

The interaction with DNAJB family members is particularly significant as these proteins function as co-chaperones that regulate the ATPase activity of HSP70 family proteins, potentially modulating HSPA4's chaperone function in ways that could promote tumor cell survival under stress conditions.

What methodological approaches should be used to study HSPA4's role in immune cell infiltration?

Studying HSPA4's role in immune cell infiltration requires sophisticated methodological approaches that can capture the complex interactions between tumor cells and immune components. Based on recent research, the following approaches are recommended:

• Comprehensive immune profiling: Utilize computational methods like CIBERSORT or ESTIMATE algorithms to assess the relationship between HSPA4 expression and various immune cell populations . This provides a broad overview of potential correlations.

• Spatial transcriptomics: Employ technologies that preserve spatial information about gene expression to understand how HSPA4-expressing tumor cells interact with immune cells in the tumor microenvironment.

• Single-cell RNA sequencing: Apply scRNA-seq to dissect the heterogeneity of immune populations in relation to HSPA4 expression levels, revealing cell-type-specific responses.

• Functional validation experiments: Design in vitro co-culture systems where HSPA4 expression in tumor cells is modulated (via knockdown or overexpression), followed by assessment of immune cell migration, activation, and cytokine production.

• In vivo models with immune monitoring: Develop mouse models with HSPA4-modulated tumors and monitor immune infiltration using flow cytometry, immunohistochemistry, and cytokine profiling.

These approaches should be integrated with immune score analyses that evaluate stromal score, immune score, and ESTIMATE score as demonstrated in previous research . When HSPA4 was highly expressed, all these scores were significantly lower (P < 0.001), suggesting reduced immune infiltration and potentially an immunosuppressive microenvironment .

What are the critical factors for successful western blotting with HSPA4 antibodies?

Western blotting with HSPA4 antibodies requires careful optimization to achieve reliable and reproducible results. Based on validated protocols, researchers should consider the following critical factors:

• Antibody selection and dilution: Select well-validated antibodies like clone AbD23625_hIgG1 and optimize dilutions within the recommended range (1/50 to 1/800) . Begin with mid-range dilutions and adjust based on signal intensity.

• Sample preparation: HSPA4 has a predicted molecular weight of ~94 kDa but appears at approximately 110 kDa in western blots, likely due to post-translational modifications . Ensure complete protein denaturation and use reducing conditions to obtain consistent banding patterns.

• Positive controls: Include positive control lysates known to express HSPA4, such as MCF7 cells, which reliably show a band at approximately 110 kDa .

• Negative controls: Implement appropriate negative controls, including HSPA4 knockdown samples or cell lines with minimal HSPA4 expression, to confirm specificity.

• Detection system optimization: Given the potential variability in HSPA4 expression levels across samples, optimize exposure times and consider using enhanced chemiluminescence (ECL) systems with appropriate sensitivity.

• Membrane blocking: Use 5% non-fat dry milk or bovine serum albumin (BSA) in Tris-buffered saline with 0.1% Tween-20 (TBST) for effective blocking to minimize background.

Following these guidelines will help ensure specific detection of HSPA4 and facilitate accurate quantification of expression differences between experimental conditions.

How can researchers optimize HSPA4 knockdown experiments to investigate functional roles?

Knockdown experiments are essential for investigating HSPA4's functional roles in cancer biology. Based on successful approaches in the literature, researchers should consider the following optimization strategies:

• siRNA design and validation: Design multiple siRNA sequences targeting different regions of HSPA4 mRNA and validate knockdown efficiency using qRT-PCR before proceeding with functional assays . The most effective siRNAs should achieve at least 70-80% reduction in HSPA4 expression.

• Transfection optimization: Optimize transfection conditions (reagent concentration, cell density, incubation time) for each cell line to maximize knockdown efficiency while minimizing cytotoxicity. For melanoma studies, the A-375 cell line has been successfully used for HSPA4 knockdown experiments .

• Time-course experiments: HSPA4 knockdown effects on cell proliferation may not be immediately apparent but become significant over time. Design experiments to assess phenotypic changes at multiple time points (e.g., 1, 2, 3, and 4 days post-transfection) .

• Appropriate functional assays: Select assays that align with HSPA4's known functions. For proliferation, the Cell Counting Kit-8 (CCK8) assay has proven effective in detecting significant differences between HSPA4 knockdown and control cells .

• Rescue experiments: Include rescue experiments where HSPA4 expression is restored in knockdown cells to confirm that observed phenotypes are specifically due to HSPA4 depletion rather than off-target effects.

In published studies, significant inhibition of tumor cell growth was observed in A-375 melanoma cells following HSPA4 knockdown, with statistically significant differences becoming evident by day 4 (P < 0.05) . This highlights the importance of conducting time-course experiments when investigating HSPA4's role in cell proliferation.

How might HSPA4 function as a potential diagnostic and therapeutic target in cancer?

HSPA4's potential as a diagnostic and therapeutic target in cancer, particularly in cutaneous malignant melanoma, is supported by multiple lines of evidence. As a diagnostic marker, HSPA4 demonstrates excellent ability to distinguish between tumor and normal tissue, with receiver operating characteristic (ROC) curve analysis showing good accuracy and sensitivity (AUC = 0.812) . This diagnostic potential is further supported by immunohistochemistry data showing positive HSPA4 staining in the majority of CMM samples compared to negative staining in normal melanocytes .

For therapeutic targeting, several promising approaches deserve investigation:

• Direct inhibition strategies: Developing small molecule inhibitors or peptide antagonists that specifically target HSPA4's ATPase domain or substrate-binding domain to disrupt its chaperone function in cancer cells.

• Exploiting synthetic lethality: Identifying pathways that become essential in HSPA4-overexpressing tumors and targeting these dependencies with existing drugs.

• Immunomodulatory approaches: Given HSPA4's negative correlation with immune cell infiltration , combining HSPA4 inhibition with immune checkpoint blockade might enhance anti-tumor immune responses.

• Targeting protein-protein interactions: Disrupting HSPA4's interactions with co-chaperones like DNAJB1, which shows positive correlation with HSPA4 expression , could impair the protein folding machinery cancer cells rely on.

• Epigenetic modulation: Since specific CpG sites in the HSPA4 gene show correlation with prognosis , epigenetic therapies might offer a way to modulate HSPA4 expression.

Research into these approaches should be guided by HSPA4's established role in promoting cell proliferation, as demonstrated by knockdown experiments showing significant inhibition of tumor cell growth , and its potential involvement in immune suppression and immune escape within the tumor microenvironment .

What is the significance of HSPA4 gene alterations and methylation patterns in cancer?

Genetic and epigenetic alterations of HSPA4 represent an important but understudied aspect of its role in cancer biology. Analysis of cancer genomics databases has revealed that approximately 3% of cutaneous malignant melanoma patients harbor HSPA4 gene alterations . While this percentage is relatively modest, it suggests that genetic alterations in HSPA4 may contribute to cancer pathogenesis in a subset of patients.

Researchers investigating HSPA4 methylation should consider:

• The relationship between methylation patterns and gene expression levels

• Whether specific methylation signatures correlate with clinical parameters beyond survival

• The potential for methylation-based biomarkers as complementary or alternative approaches to expression-based biomarkers

• Whether methylation-modifying therapies could normalize HSPA4 expression in cancer cells

The identification of prognostically significant CpG sites in the HSPA4 gene opens new avenues for biomarker development and potentially targeted epigenetic therapies that might restore normal HSPA4 expression patterns in cancer cells.

What are the most promising future directions for HSPA4 monoclonal antibody research?

The future of HSPA4 monoclonal antibody research holds significant promise across multiple fronts. Based on current evidence, the most promising directions include:

• Expanding diagnostic applications: Developing standardized immunohistochemistry protocols using validated HSPA4 antibodies for routine clinical assessment of cancer tissues, particularly in melanoma where HSPA4 shows strong diagnostic value (AUC = 0.812) .

• Therapeutic antibody development: Exploring the potential of HSPA4-targeting antibodies for cancer therapy, particularly antibody-drug conjugates that could deliver cytotoxic payloads specifically to HSPA4-overexpressing tumor cells.

• Combination biomarker strategies: Investigating how HSPA4 expression might complement other biomarkers to improve diagnostic accuracy and prognostic prediction in cancer, potentially as part of multi-protein panels.

• Mechanistic investigations: Using highly specific monoclonal antibodies to further elucidate HSPA4's interactions with other proteins, particularly DNAJB1 and DNAJB6 , and how these interactions influence cancer cell biology.

• Immune modulation research: Exploring HSPA4's relationship with immune cell infiltration and function could reveal new insights into cancer immunotherapy responses and resistance mechanisms.