HSPA12B Antibody

What is HSPA12B and where is it predominantly expressed?

HSPA12B (Heat shock protein A12B) is the newest member of the HSP70 family of proteins, which plays a considerable protective role for cells, tissues, and organs against various noxious conditions . HSPA12B is predominantly expressed in vascular endothelium . Expression analysis using Northern blot, in situ hybridization, and immunostaining with HSPA12B-specific antibodies has demonstrated that HSPA12B is highly specific to endothelial cells, particularly human umbilical vein endothelial cells (HUVECs) . Comparative studies using real-time PCR have shown that HSPA12B expression is approximately 26-fold higher in HUVECs than in other cell types, including fibrosarcoma cells (HT1080), human embryonic kidney epithelial cells (HEK 293), human colon cancer cells (DLD1), ovarian cancer cells (OVCAR3), and podocytes .

What are the recommended applications for HSPA12B antibodies in research?

HSPA12B antibodies can be effectively utilized in multiple experimental techniques, including:

• Immunohistochemistry (IHC): Typically used at dilutions of 1:50-1:200 to detect HSPA12B expression in tissue samples . This technique has been successfully employed to examine HSPA12B expression in NHL and non-tumor tissue samples .

• Western blotting: Recommended concentration range of 0.04-0.4 μg/mL for protein detection . This application is useful for quantifying HSPA12B expression levels in various cell types and under different experimental conditions.

• Immunofluorescence: Used to map HSPA12B at the subcellular level as part of efforts like the Human Protein Atlas project .

• Functional studies: HSPA12B-neutralizing antibodies have been used in angiogenesis assays (Matrigel) and migration assays to investigate the protein's role in endothelial cell function .

How should HSPA12B antibodies be stored and handled for optimal results?

For optimal performance, HSPA12B antibodies should be:

• Stored at -20°C when not in use .

• Shipped on wet ice to maintain antibody integrity .

• Maintained in buffered aqueous glycerol solution to preserve antibody function .

• Aliquoted to avoid repeated freeze-thaw cycles that may compromise antibody performance.

• Validated for specificity prior to experimental use through appropriate controls.

What are the optimal methods for detecting HSPA12B expression in tissue samples?

For detecting HSPA12B expression in tissue samples, immunohistochemistry (IHC) has proven to be particularly effective. A standardized protocol includes:

• Tissue preparation: Fixation in formalin and embedding in paraffin, followed by sectioning at 4-5 μm thickness.

• Antigen retrieval: Usually performed using citrate buffer (pH 6.0) at high temperature (95-100°C) for 15-20 minutes.

• Blocking and antibody incubation:

  • Block endogenous peroxidase activity with 3% H₂O₂

  • Block non-specific binding with appropriate serum

  • Incubate with anti-HSPA12B antibody at optimized dilution (typically 1:50-1:200)

  • Incubate with secondary antibody and detection system

• Controls: PBS-based controls should be included to evaluate the specificity of IHC signaling .

This methodology has successfully distinguished HSPA12B expression between NHL samples and reactive lymphadenopathy tissue samples, revealing that HSPA12B expression is lower in NHL samples compared to non-tumor tissue .

How can HSPA12B antibodies be used to investigate protein function in angiogenesis?

To investigate HSPA12B's role in angiogenesis, researchers can employ the following methodological approaches:

These methods have demonstrated that in vitro angiogenesis and migration are inhibited in HUVECs in the presence of HSPA12B-neutralizing antibodies, supporting HSPA12B's role as a regulator of angiogenesis .

What techniques can be used to study HSPA12B's role in inflammatory responses?

To investigate HSPA12B's role in inflammatory responses, particularly in endothelial cells, researchers can implement these methodological approaches:

• Permeability assay:

  • Culture endothelial cell monolayers on permeable supports

  • Treat with lipopolysaccharide (LPS) with or without HSPA12B overexpression

  • Measure permeability using fluorescent-labeled dextran

• Polymorphonuclear leukocyte (PMN) adhesion assay:

  • Treat endothelial cells with LPS with or without HSPA12B manipulation

  • Add fluorescently-labeled PMNs and allow adhesion

  • Wash and quantify adherent PMNs

• Inflammatory marker analysis:

  • Measure adhesion molecules (ICAM-1, VCAM-1, E-selectin) by RT-qPCR and Western blot

  • Quantify inflammatory cytokine production (IL-6, TNF-α) by ELISA

• Signaling pathway investigation:

  • Evaluate PI3K/Akt pathway activation through Western blot

  • Use pathway inhibitors (e.g., Wortmannin) to confirm mechanism

These techniques have revealed that HSPA12B suppresses LPS-induced HUVEC permeability, reduces PMN adhesion to HUVECs, and inhibits LPS-induced upregulation of adhesion molecules and inflammatory cytokines through activation of the PI3K/Akt signaling pathway .

How does HSPA12B expression correlate with non-Hodgkin's lymphoma (NHL) variants and prognosis?

HSPA12B expression shows distinct patterns across NHL variants with significant prognostic implications:

Statistical analysis shows a highly significant difference in HSPA12B expression between NHL and normal lymph tissue (P<0.05) . Survival analysis using Kaplan-Meier curves and log-rank tests has confirmed correlation between HSPA12B expression levels and patient outcomes, suggesting that HSPA12B can potentially serve as both a predictor of NHL prognosis and therapeutic effect .

What is the relationship between HSPA12B and cell adhesion-mediated drug resistance (CAM-DR) in NHL?

HSPA12B plays a critical role in cell adhesion-mediated drug resistance in NHL through several mechanisms:

• Induction of HSPA12B expression:

  • HSPA12B expression is induced when NHL cells adhere to fibronectin (FN) or bone marrow stroma cells (BMSCs) .

  • This suggests that the cellular microenvironment regulates HSPA12B expression.

• Anti-apoptotic function:

  • Overexpression of HSPA12B in NHL cell lines (OCI-LY8 and Daudi) resulted in:

    • Increased cell viability when treated with doxorubicin (0.5 μmol/L)

    • Up-regulation of anti-apoptotic protein Bcl-2

    • Down-regulation of pro-apoptotic protein Bax

    • Significant decrease in Annexin V-positive cells (indicating reduced apoptosis)

• Enhancement of CAM-DR:

  • HSPA12B overexpression enhances resistance to chemotherapy drugs when NHL cells are adhered to extracellular matrix components or stromal cells .

  • This finding suggests that HSPA12B may be a potential novel target for overcoming drug resistance in NHL treatment.

How can HSPA12B antibodies be used to study its role in atherosclerosis?

HSPA12B is enriched in atherosclerotic lesions, suggesting a potential role in this pathology . Researchers investigating HSPA12B in atherosclerosis can employ the following approaches:

• Immunohistochemical analysis:

  • Use anti-HSPA12B antibodies to detect expression patterns in:

    • Atherosclerotic plaques compared to normal vessel walls

    • Different stages of atherosclerotic lesion development

    • Various cell types within lesions (endothelial cells, macrophages, smooth muscle cells)

• Functional studies in endothelial cells:

  • Manipulate HSPA12B expression (overexpression or knockdown)

  • Assess effects on:

    • Endothelial activation markers (adhesion molecules, cytokines)

    • Monocyte adhesion to endothelial cells

    • Endothelial permeability under pro-atherogenic conditions

• Interaction studies:

  • Employ yeast two-hybrid screening to identify HSPA12B-interacting proteins relevant to atherosclerosis

  • Confirm interactions through co-immunoprecipitation using anti-HSPA12B antibodies

  • Characterize functional significance of these interactions

These methodologies can help elucidate how HSPA12B might influence atherosclerosis development through its effects on endothelial function, inflammation, and angiogenesis.

What are the optimal strategies for generating and validating HSPA12B antibodies for research?

Generating and validating high-quality HSPA12B antibodies requires careful consideration of several methodological aspects:

• Antibody generation strategies:

  • Polyclonal antibody generation: Immunize rabbits with synthesized peptides derived from HSPA12B, such as the sequence "QLLDLSGRAPGGGRLGERRSIDSSFRQAREQLRRSRHSRTFLVESGVGELWAEMQAGDRYVVA" .

  • Select immunogenic epitopes based on computational prediction of surface accessibility and uniqueness.

  • Purify antibodies through affinity chromatography to enhance specificity.

• Validation methods:

  • Western blot analysis against recombinant HSPA12B and endothelial cell lysates

  • Immunoprecipitation followed by mass spectrometry

  • Immunohistochemistry on tissues with known HSPA12B expression patterns

  • Testing on samples from HSPA12B knockout models (negative controls)

  • Peptide competition assays to confirm specificity

• Cross-reactivity assessment:

  • Test against related proteins, particularly HSPA12A which is closely related to HSPA12B

  • Evaluate species cross-reactivity (human, mouse, rat) to determine versatility for various research models

These rigorous validation steps ensure the development of reliable antibodies for studying HSPA12B in various research contexts.

How can contradictory findings about HSPA12B expression in different cancer types be reconciled?

The expression and function of HSPA12B in cancer biology remains controversial . To reconcile contradictory findings, researchers should consider:

• Methodological differences:

  • Detection methods: Compare results obtained through different techniques (IHC, Western blot, RT-qPCR)

  • Antibody specificity: Evaluate whether different antibodies target different epitopes

  • Sample preparation: Assess how tissue fixation and processing might affect detection

• Cancer-specific contexts:

  • Cell lineage effects: HSPA12B expression appears to be cell-type specific, predominantly found in endothelial cells

  • In NHL, expression is higher in indolent lymphomas compared to aggressive variants

  • The role may differ between tumors of endothelial origin versus those where HSPA12B's effect is mediated through tumor microenvironment

• Experimental design for resolving contradictions:

  • Parallel analysis of multiple cancer types using identical methods

  • Combined analysis of tumor cells and associated vasculature

  • Correlation with angiogenesis markers

  • Functional studies in relevant cell types

• Dual functions analysis:

  • Investigate whether HSPA12B may have context-dependent pro- or anti-tumorigenic effects

  • Assess whether effects on angiogenesis versus direct effects on tumor cells might explain contradictory findings

A comprehensive approach incorporating these considerations can help resolve apparent contradictions and clarify HSPA12B's true role in different cancer contexts.

What experimental approaches can be used to investigate the mechanisms by which HSPA12B regulates angiogenesis?

To elucidate the molecular mechanisms through which HSPA12B regulates angiogenesis, researchers can employ these advanced experimental approaches:

• Protein interaction studies:

  • Yeast two-hybrid screening has identified that HSPA12B interacts with multiple proteins involved in angiogenesis regulation

  • Co-immunoprecipitation using anti-HSPA12B antibodies can confirm these interactions in endothelial cells

  • Proximity ligation assays can visualize interactions in situ

• Signaling pathway analysis:

  • Investigate the PI3K/Akt pathway, which has been implicated in HSPA12B's anti-inflammatory effects

  • Use pathway-specific inhibitors (e.g., Wortmannin) to determine causality

  • Perform phosphoproteomic analysis to identify additional regulated pathways

• Transcriptional regulation:

  • ChIP-seq to identify genes directly regulated by HSPA12B or its interacting partners

  • RNA-seq in HSPA12B overexpression or knockdown models to identify global transcriptional changes

  • Analysis of angiogenesis-related gene expression networks

• In vivo models:

  • Transgenic mice expressing Enhanced-Green-Fluorescent-Protein under the control of the HSPA12B promoter to track expression during angiogenesis

  • Conditional HSPA12B knockout models specific to endothelial cells

  • Angiogenesis assays (retinal angiogenesis, tumor angiogenesis, wound healing) in these models

• Structural biology:

  • Analysis of HSPA12B's ATP-binding domain and substrate-binding domain

  • Investigation of how these domains might regulate interactions with angiogenesis-related proteins

These approaches can provide mechanistic insights into how HSPA12B functions as a novel regulator of angiogenesis and potentially inform therapeutic strategies targeting angiogenesis-related diseases.

How might HSPA12B antibodies be utilized in diagnostic or prognostic applications for NHL?

Based on research findings, HSPA12B antibodies show potential for diagnostic and prognostic applications in NHL:

• Diagnostic applications:

  • Differential diagnosis: HSPA12B expression levels can help distinguish between aggressive and indolent NHL variants

  • Immunohistochemical panels: Including anti-HSPA12B antibodies alongside established markers may enhance diagnostic accuracy

  • Expression pattern analysis: The distribution pattern of HSPA12B within lymphoma tissue provides additional diagnostic information

• Prognostic stratification:

  • Survival prediction: Statistical analysis using Kaplan-Meier curves has demonstrated correlation between HSPA12B expression levels and patient outcomes

  • Risk stratification: HSPA12B expression could be incorporated into prognostic scoring systems

  • Multivariate analysis using Cox's proportional hazards model has shown HSPA12B as an independent prognostic factor

• Treatment response prediction:

  • CAM-DR prediction: Since HSPA12B is implicated in cell adhesion-mediated drug resistance, its expression levels might predict response to conventional chemotherapy

  • HSPA12B might serve as a predictor of therapeutic effect, potentially identifying patients who would benefit from combination therapies targeting drug resistance mechanisms

What considerations should researchers take into account when designing therapeutic strategies targeting HSPA12B?

When considering HSPA12B as a therapeutic target, researchers should address these key factors:

• Cell type specificity:

  • HSPA12B is predominantly expressed in endothelial cells

  • Targeting strategies must consider potential effects on normal vasculature

  • Delivery systems may need to differentiate between tumor-associated endothelium and normal endothelium

• Dual roles in different contexts:

  • Anti-inflammatory effects in endothelial cells

  • Anti-apoptotic effects in NHL cells

  • Angiogenesis promotion

  These multiple functions necessitate context-specific targeting approaches.

• Targeting approaches:

  • Direct inhibition: HSPA12B-neutralizing antibodies have shown efficacy in blocking angiogenesis

  • Indirect modulation: Targeting pathways regulated by HSPA12B, such as PI3K/Akt

  • Combination strategies: Co-targeting HSPA12B and adhesion molecules to overcome CAM-DR in NHL

• Potential applications:

  • NHL treatment: Targeting HSPA12B to overcome drug resistance mechanisms

  • Anti-angiogenic therapy: Inhibiting HSPA12B to reduce pathological angiogenesis

  • Vascular inflammation: Enhancing HSPA12B to reduce inflammatory responses in endothelial cells

These considerations highlight the complex nature of HSPA12B as a therapeutic target and emphasize the need for careful context-specific approaches.

What are common technical challenges when using HSPA12B antibodies and how can they be overcome?

Researchers working with HSPA12B antibodies may encounter several technical challenges:

• Background staining in immunohistochemistry:

  • Challenge: Non-specific binding leading to high background

  • Solutions:

    • Optimize blocking conditions (use 3-5% BSA or normal serum)

    • Increase washing steps and duration

    • Titrate antibody concentration (start with recommended 1:50-1:200 dilution)

    • Include PBS-based controls to evaluate specificity

• Variability in staining intensity:

  • Challenge: Inconsistent results between experiments

  • Solutions:

    • Standardize tissue fixation and processing

    • Use automated staining platforms when available

    • Include positive control tissues (endothelial cells) in each experiment

    • Develop quantitative scoring systems

• Cross-reactivity with HSPA12A:

  • Challenge: HSPA12A is closely related to HSPA12B

  • Solutions:

    • Select antibodies raised against unique epitopes

    • Verify specificity using tissues from HSPA12B knockout models

    • Perform parallel staining with HSPA12A-specific antibodies to compare patterns

• Detection in cell types with low expression:

  • Challenge: HSPA12B is predominantly expressed in endothelial cells

  • Solutions:

    • Use more sensitive detection systems (e.g., tyramide signal amplification)

    • Consider more sensitive techniques like RT-qPCR for quantification

    • Enrich for cell populations of interest before analysis

Implementing these solutions can help overcome common technical challenges and ensure reliable results when working with HSPA12B antibodies.

How can researchers optimize HSPA12B antibody use for detecting changes in expression levels under different experimental conditions?

To optimize detection of HSPA12B expression changes across experimental conditions:

• Quantitative Western blot optimization:

  • Use standardized loading controls (β-actin, GAPDH, or vinculin)

  • Establish linear detection range for HSPA12B signal

  • Consider dual-color detection systems to simultaneously visualize HSPA12B and loading controls

  • Use appropriate positive controls (HUVEC lysates) and negative controls

  • Recommended antibody concentration: 0.04-0.4 μg/mL

• RT-qPCR optimization:

  • Design primers spanning exon-exon junctions to avoid genomic DNA amplification

  • Validate primer efficiency using standard curves

  • Select appropriate reference genes for specific experimental conditions

  • Use multiple reference genes for normalization

  • Compare relative expression using the 2^(-ΔΔCt) method

• Immunohistochemistry/immunofluorescence quantification:

  • Standardize image acquisition parameters

  • Use digital image analysis software for objective quantification

  • Consider both staining intensity and proportion of positive cells

  • Develop scoring systems (e.g., H-score or Allred score)

  • Blind scoring to experimental conditions to reduce bias

• Flow cytometry for cellular HSPA12B detection:

  • Optimize cell permeabilization protocols for intracellular staining

  • Use fluorescence minus one (FMO) controls to set gates

  • Consider median fluorescence intensity (MFI) for quantitative comparisons

  • Analyze shifts in expression across experimental conditions using histogram overlays