DNAJB4 Antibody

What is DNAJB4 and why is it a significant research target?

DNAJB4 (DnaJ Heat Shock Protein Family (Hsp40) Member B4) functions as a molecular chaperone that regulates protein homeostasis. It belongs to the HSP40 family and transfers substrates to Hsp70, stimulating its ATPase domain and conferring specificity to this chaperone family. DNAJB4 has emerged as a significant research target due to its roles in cancer progression and protein quality control. It has been shown to suppress cancer progression, particularly in breast cancer, making it an important focus for oncology research .

What types of DNAJB4 antibodies are commercially available for research?

Several types of DNAJB4 antibodies are commercially available for research applications:

These antibodies are generally supplied in liquid form with storage buffers containing PBS, sodium azide, glycerol, and sometimes BSA .

What are the optimal sample preparation techniques for detecting DNAJB4 in different tissue types?

For optimal detection of DNAJB4 in tissue samples, researchers should:

• For paraffin-embedded tissues: Use formalin fixation followed by standard paraffin embedding protocols. Antigen retrieval with TE buffer (pH 9.0) is recommended, though citrate buffer (pH 6.0) may also be effective .

• For cell culture samples: PFA fixation followed by Triton X-100 permeabilization has been successfully used for immunofluorescence applications .

• For protein extraction: Standard lysis buffers containing protease inhibitors are effective for extracting DNAJB4 from various cell lines including HeLa, MCF-7, RT4, and tissue samples like mouse skeletal muscle .

The choice of sample preparation should be guided by the specific application and tissue type being studied .

How should researchers optimize antibody dilutions for different DNAJB4 detection applications?

Optimization of DNAJB4 antibody dilutions varies by application and specific antibody:

It's critical to conduct preliminary experiments with positive controls (e.g., HeLa cells, human pancreas tissue) to determine optimal conditions for each specific experimental system .

What strategies can address inconsistent DNAJB4 detection in cancer tissue samples?

When facing inconsistent DNAJB4 detection in cancer tissues, consider these approaches:

• Implement dual validation methods by combining techniques (e.g., IHC with Western blot)

• Account for variable DNAJB4 expression across cancer types and stages, as DNAJB4 is significantly downregulated in multiple cancers including breast cancer, bladder urothelial carcinoma, and colon adenocarcinoma

• Optimize antigen retrieval methods specifically for each tissue type

• Consider cellular localization patterns, as DNAJB4 detection may vary depending on subcellular distribution

• Use multiple antibodies targeting different epitopes of DNAJB4 to confirm expression patterns

• Include appropriate positive controls such as normal tissues with known DNAJB4 expression patterns (e.g., thyroid, testis tissues)

These strategies can help overcome the challenges associated with DNAJB4's variable expression patterns in cancer tissues .

How should DNAJB4 antibodies be incorporated into studies investigating its tumor suppressor function?

When designing experiments to investigate DNAJB4's tumor suppressor function:

• Combine DNAJB4 antibody-based detection with functional assays:

  • Proliferation assays (e.g., CCK-8, colony-forming)

  • Migration assays (e.g., wound healing)

  • In vivo xenograft models

• Incorporate pathway analysis:

  • Use antibodies against Hippo pathway components alongside DNAJB4 antibodies

  • Analyze epithelial-mesenchymal transition (EMT) markers

• Design comparative studies:

  • Compare DNAJB4 expression between normal and cancer tissues

  • Analyze correlation between DNAJB4 expression and clinical outcomes

• Consider immune infiltration analysis:

  • Pair DNAJB4 detection with CD4+, CD8+ T cell markers and PD-L1

  • Evaluate the impact of DNAJB4 overexpression on immune cell recruitment

These approaches have successfully demonstrated DNAJB4's role in suppressing breast cancer progression and promoting tumor immunogenicity through the Hippo pathway .

What controls should be included when validating DNAJB4 antibody specificity for research applications?

For rigorous validation of DNAJB4 antibody specificity:

• Positive control samples:

  • Cell lines with confirmed DNAJB4 expression (HeLa, MCF-10A)

  • Normal tissues with known DNAJB4 expression (pancreas, thyroid)

• Negative controls:

  • DNAJB4 knockdown/knockout cells using siRNA or CRISPR-Cas9

  • Secondary antibody-only controls to assess background

  • Blocking peptide competition assays

• Cross-reactivity assessment:

  • Testing against recombinant proteins from the same family

  • Comparing results from multiple antibodies targeting different DNAJB4 epitopes

• Application-specific controls:

  • For Western blot: molecular weight verification (38 kDa)

  • For IHC/IF: peptide competition and isotype controls

These comprehensive validation approaches ensure that experimental findings accurately reflect DNAJB4 biology rather than non-specific artifacts .

How should researchers interpret variable DNAJB4 expression patterns across different cancer subtypes?

When interpreting variable DNAJB4 expression patterns:

• Consider context-specific functions:

  • DNAJB4 shows significantly reduced expression across multiple cancer types (breast cancer, bladder urothelial carcinoma, colon adenocarcinoma, kidney chromophobe)

  • Low DNAJB4 expression correlates with poor prognosis in breast cancer patients, specifically affecting distant metastasis-free survival (DMFS) and recurrence-free survival (RFS)

• Account for molecular subtype variation:

  • Expression patterns may differ between cancer subtypes

  • Integrate bioinformatic analysis with experimental validation

• Correlate with clinical parameters:

  • Analyze DNAJB4 expression in relation to tumor stage, grade, and patient outcomes

  • Consider multivariate analysis to identify confounding factors

• Contextualize findings within known DNAJB4 mechanisms:

  • Inhibition of cell proliferation and migration through Hippo pathway activation

  • Modulation of immune infiltration and PD-L1 expression

This comprehensive approach allows for more nuanced interpretation of DNAJB4's role across cancer types .

What molecular mechanisms explain DNAJB4 regulation and how can researchers investigate them?

To investigate DNAJB4 regulatory mechanisms:

• Transcriptional regulation:

  • Examine promoter activity using reporter assays

  • Identify transcription factors using ChIP assays

• Post-transcriptional regulation:

  • Investigate microRNA interactions, particularly hsa-miR-183-5p which targets DNAJB4

  • Analyze mRNA stability using actinomycin D chase experiments

• Epitranscriptomic regulation:

  • Study m6A modifications in DNAJB4 mRNA 5'-UTR using SELECT (single-base elongation and ligation-based qPCR amplification)

  • Investigate how HSP90 inhibitors stimulate DNAJB4 protein expression through m6A modifications

• Post-translational regulation:

  • Analyze protein stability using cycloheximide chase assays

  • Investigate potential ubiquitination or other modifications

• Stress-responsive regulation:

  • Examine DNAJB4 expression changes in response to HSP90 inhibitors (e.g., ganetespib)

  • Analyze stress-induced translational control

These approaches have revealed that DNAJB4 expression is regulated at multiple levels, including through m6A modifications that influence translation efficiency .

How can researchers address non-specific binding when using DNAJB4 antibodies in multiplexed immunofluorescence?

To address non-specific binding in multiplexed immunofluorescence:

• Optimize blocking conditions:

  • Test different blocking agents (BSA, normal serum, commercial blockers)

  • Extend blocking time to reduce background

• Antibody titration:

  • Test dilution ranges from 1:50-1:500 for immunofluorescence applications

  • Use positive control cells with known DNAJB4 expression (e.g., MCF-7, A-431)

• Sequential staining approach:

  • Apply antibodies sequentially rather than simultaneously

  • Include washing steps with detergent between antibody applications

• Cross-reactivity mitigation:

  • Use antibody fragments or monovalent formats when available

  • Pre-adsorb antibodies with tissue/cell lysates

• Spectral unmixing:

  • Apply computational approaches to separate overlapping signals

  • Use appropriate fluorophores with minimal spectral overlap

These approaches can significantly improve specificity when using DNAJB4 antibodies in complex multiplexed immunofluorescence protocols .

What strategies can improve detection sensitivity for low DNAJB4 expression in clinical samples?

For improved detection of low DNAJB4 expression:

• Signal amplification techniques:

  • Tyramide signal amplification (TSA) for IHC/IF applications

  • Enhanced chemiluminescence (ECL) substrates for Western blot

• Sample enrichment:

  • Immunoprecipitation before Western blot analysis

  • Use of tissue microarrays for standardized IHC detection

• Optimized antigen retrieval:

  • Test both TE buffer (pH 9.0) and citrate buffer (pH 6.0)

  • Optimize duration and temperature for specific tissue types

• Alternative detection platforms:

  • Digital PCR for mRNA quantification

  • Proximity ligation assay for protein detection

• Enhanced imaging and quantification:

  • Digital image analysis algorithms

  • Whole slide scanning for comprehensive tissue evaluation

These approaches have successfully detected DNAJB4 even in cancer samples with reduced expression .

How can DNAJB4 antibodies be used to investigate interactions with the Hippo signaling pathway in cancer?

To investigate DNAJB4-Hippo pathway interactions:

• Co-immunoprecipitation approaches:

  • Use DNAJB4 antibodies to pull down complexes and probe for Hippo pathway components

  • Perform reciprocal IP with antibodies against Hippo pathway proteins (YAP, TAZ, LATS)

• Proximity ligation assays:

  • Detect in situ interactions between DNAJB4 and Hippo pathway components

  • Visualize subcellular localization of interaction sites

• Functional validation studies:

  • Combine DNAJB4 overexpression with LATS inhibition (e.g., LATS-IN-1)

  • Analyze downstream effects on YAP/TAZ nuclear localization and target gene expression

• In vivo models:

  • Use DNAJB4 antibodies to monitor expression in xenograft models treated with Hippo pathway modulators

  • Correlate DNAJB4 expression with CD4+, CD8+ T cell infiltration and PD-L1 levels

These approaches have revealed that DNAJB4 activates the Hippo pathway, leading to inhibition of cell proliferation and migration, and enhanced anti-tumor immunity .

What emerging technologies can enhance DNAJB4 detection in spatial transcriptomics and proteomics?

Emerging technologies for advanced DNAJB4 analysis:

• Spatial proteomics applications:

  • Imaging mass cytometry for multiplexed protein detection with spatial resolution

  • Digital spatial profiling to quantify DNAJB4 in precise tissue regions

  • CODEX (CO-Detection by indEXing) for highly multiplexed tissue imaging

• Combined spatial transcriptomics:

  • Visium spatial transcriptomics to correlate DNAJB4 mRNA distribution with protein localization

  • MERFISH or seqFISH for single-cell spatial resolution of DNAJB4 mRNA

• Advanced microscopy techniques:

  • Super-resolution microscopy to analyze DNAJB4 subcellular localization

  • Live-cell imaging with tagged DNAJB4 to monitor dynamic changes

• Artificial intelligence integration:

  • Machine learning algorithms for automated quantification of DNAJB4 expression patterns

  • Deep learning approaches for correlating DNAJB4 expression with histopathological features

These technologies would significantly enhance our understanding of DNAJB4's spatial distribution and functional interactions in complex tissues .

How can researchers investigate epitranscriptomic regulation of DNAJB4 using specialized antibodies?

To study epitranscriptomic regulation of DNAJB4:

• m6A-focused approaches:

  • Use m6A-specific antibodies for MeRIP-seq to map modification sites in DNAJB4 mRNA

  • Apply SELECT (single-base elongation and ligation-based qPCR amplification) to quantify m6A at specific sites in the 5'-UTR

• Writer/eraser/reader protein analysis:

  • Investigate interactions between DNAJB4 mRNA and m6A machinery components

  • Employ RNA immunoprecipitation with antibodies against writer (METTL3/14), eraser (FTO, ALKBH5), and reader proteins

• Translational efficiency measurement:

  • Conduct polysome fractionation followed by RT-qPCR to assess DNAJB4 mRNA translation

  • Use puromycin incorporation assays to measure nascent DNAJB4 protein synthesis

• Stress response studies:

  • Analyze how HSP90 inhibitors (e.g., ganetespib) alter m6A modification and DNAJB4 expression

  • Investigate the relationship between heat shock response and epitranscriptomic regulation

These approaches have revealed that HSP90 inhibitors stimulate DNAJB4 protein expression partly through an epitranscriptomic mechanism involving m6A modifications in the 5'-UTR .

How do different DNAJB4 antibodies compare in terms of specificity and sensitivity across applications?

Comparative analysis of DNAJB4 antibodies:

When selecting between these antibodies, researchers should consider:

• Target application (some perform better in specific applications)

• Host animal compatibility with experimental design

• Validated reactivity with species of interest

• Specific epitope recognition and potential cross-reactivity

This comparison helps researchers select the most appropriate antibody for their specific experimental requirements .

What methodological approaches can resolve contradictory findings when studying DNAJB4 expression in different experimental systems?

To resolve contradictory findings in DNAJB4 research:

• Standardize detection methods:

  • Use consistent antibody clones and dilutions across studies

  • Standardize sample preparation protocols

  • Implement quantitative analysis methods

• Control for biological variables:

  • Account for cell density and growth conditions that may affect DNAJB4 expression

  • Consider tissue heterogeneity in complex samples

  • Document passage number of cell lines

• Apply orthogonal validation:

  • Combine protein detection with mRNA analysis

  • Use multiple antibodies targeting different DNAJB4 epitopes

  • Implement genetic approaches (siRNA, CRISPR) for validation

• Context-specific analysis:

  • Consider stress conditions that may affect DNAJB4 (HSP90 inhibitors can increase expression)

  • Account for cancer subtype-specific expression patterns

  • Evaluate epitranscriptomic modifications that may affect detection

• Statistical rigor:

  • Increase sample sizes to account for biological variability

  • Apply appropriate statistical tests with corrections for multiple comparisons

  • Report effect sizes alongside p-values

These approaches have successfully resolved apparent contradictions in DNAJB4 expression patterns across different experimental systems .

How might DNAJB4 antibodies contribute to developing cancer prognostic markers and therapeutic strategies?

DNAJB4 antibodies have significant potential in cancer research applications:

• Development of prognostic tools:

  • Standardized IHC protocols using validated DNAJB4 antibodies could assess expression in patient samples

  • Low DNAJB4 expression correlates with poor prognosis in breast cancer patients (affects distant metastasis-free survival and recurrence-free survival)

  • Integration with other biomarkers could create comprehensive prognostic panels

• Therapeutic monitoring:

  • DNAJB4 antibodies can monitor expression changes during HSP90 inhibitor treatment

  • Potential for companion diagnostic development

  • Assessment of treatment efficacy in real-time

• Immunotherapy connections:

  • DNAJB4 overexpression enhances CD4+ and CD8+ T cell infiltration and reduces PD-L1 levels in tumors

  • DNAJB4 antibodies could help identify patients likely to respond to immunotherapies

  • Monitoring DNAJB4-mediated changes in tumor immune microenvironment

• Drug development:

  • Screening compounds that induce DNAJB4 expression

  • Target validation for novel therapeutic approaches

  • Combination therapy strategies leveraging DNAJB4's tumor suppressive functions

These applications leverage DNAJB4's roles in cancer suppression and immune modulation to develop new clinical tools .

What are the most promising directions for integrating DNAJB4 research with emerging immunotherapy approaches?

Integration of DNAJB4 research with immunotherapy:

• Tumor microenvironment modulation:

  • DNAJB4 overexpression enhances CD4+ and CD8+ T cell infiltration while reducing PD-L1 levels in tumors

  • Research combining DNAJB4 expression analysis with immune checkpoint inhibitor response

  • Development of strategies to upregulate DNAJB4 to improve immunotherapy outcomes

• Combination therapy approaches:

  • HSP90 inhibitors increase DNAJB4 expression and could potentially be combined with immunotherapies

  • Investigation of DNAJB4's effects on additional immune cell populations beyond T cells

  • Exploration of synergistic effects between DNAJB4-inducing compounds and immune checkpoint blockade

• Mechanistic studies:

  • Detailed analysis of how DNAJB4 influences immune cell recruitment and function

  • Investigation of DNAJB4's role in antigen presentation and processing

  • Exploration of Hippo pathway connections to immune regulation through DNAJB4

• Translational applications:

  • Development of assays to stratify patients based on DNAJB4 expression profiles

  • Creation of ex vivo systems to test immunotherapy efficacy in relation to DNAJB4 expression

  • Design of clinical trials incorporating DNAJB4 expression as a biomarker