thbs4b Antibody

What is Thrombospondin-4B and what key cellular functions does it mediate?

Thrombospondin-4B (THBS4B) is an adhesive glycoprotein that plays a critical role in mediating cell-to-cell and cell-to-matrix interactions across multiple tissues . It is involved in various cellular processes including proliferation, migration, adhesion, and attachment . Recent research has demonstrated that THBS4 functions beyond its traditional characterization as a secreted extracellular matrix (ECM) or matricellular protein, with emerging evidence pointing to important intracellular roles, particularly within the endoplasmic reticulum (ER) . Within the ER, THBS4 directly binds to the luminal domain of ATF6α, promoting its translocation to the Golgi and subsequently to the nucleus, thereby activating genes involved in the adaptive ER stress response . This previously underappreciated intracellular function appears to be critical for certain cellular protection mechanisms, particularly in cardiac and skeletal muscle tissues.

How do researchers select the appropriate THBS4B antibody for their experimental model?

When selecting a THBS4B antibody, researchers should consider several critical factors based on their specific experimental needs. First, evaluate the species reactivity - commercial antibodies are available with reactivity to zebrafish, human, and mouse THBS4/THBS4B, among others . For cross-species studies, verify whether the antibody cross-reacts with THBS4B from other species or if it's species-specific. The zebrafish-specific THBS4B antibody (N-terminal) has been successfully used in multiple publications and is validated for Western blot and immunohistochemistry applications .

Second, consider the specific application requirements. Different antibodies are optimized for distinct techniques:

• For Western blotting: Select antibodies validated to detect the expected molecular weight band (approximately 105.9 kDa for human THBS4)

• For immunohistochemistry: Choose antibodies demonstrated to work in fixed tissues with minimal background

• For immunofluorescence: Select antibodies compatible with your fixation method and detection system

Finally, review published literature where the antibody has been successfully utilized. For example, the zebrafish THBS4B antibody has been cited in at least 4 publications, providing confidence in its reliability for certain applications .

What expression patterns of THBS4B should researchers expect in different tissue types?

Expression patterns of THBS4B vary significantly across tissue types and can be influenced by pathophysiological conditions. In normal cardiovascular tissues, THBS4 shows notable expression in heart tissue, where it has been detected at approximately 130-158 kDa using Western blot analysis . In vascular lesions, THBS4 expression is altered as demonstrated by studies using Thbs4−/− mice .

In skeletal muscle, THBS4 expression is crucial for maintaining muscle integrity and protection against muscular dystrophy . The protein appears to function both within myofibers and at specialized membrane structures.

In inflammatory conditions such as LPS-induced peritonitis, THBS4 expression increases significantly in macrophages both in the peritoneal cavity and tissue . Quantitative RT-PCR analysis revealed a fold increase over control mice injected with PBS, with statistically significant differences (p < 0.05, n = 3) .

Cultured macrophage-like cells (RAW264.7) and bone marrow-derived macrophages (BMDM) also show inducible THBS4 expression when stimulated with LPS (0.5 μg/ml), with expression detectable from 1-24 hours post-treatment by Western blotting .

What are the optimal protocols for THBS4B detection in Western blotting applications?

For optimal Western blot detection of THBS4B, researchers should follow these evidence-based methodological guidelines:

Sample Preparation and Loading:

• For heart tissue samples, prepare lysates under reducing conditions

• Load 0.2 mg/mL of tissue lysate for optimal detection

• Include appropriate positive controls (human heart tissue has shown consistent THBS4 expression)

Detection Parameters:

• Primary Antibody: For human THBS4, use 0.2-2 μg/mL of anti-THBS4 antibody (such as Goat Anti-Human Thrombospondin-4 Antigen Affinity-purified Polyclonal Antibody)

• Secondary Antibody: Use HRP-conjugated secondary antibody at manufacturer-recommended dilution (e.g., 1:50 dilution has been successful in published protocols)

• Expected Band Size: Human THBS4 typically appears at approximately 130 kDa under reducing conditions, but may show variation (130-158 kDa) depending on sample type and electrophoresis system

Optimization Considerations:

• Buffer System: Immunoblot Buffer Group 1 has been successfully used in published protocols

• Separation System: For proteins in the 105-158 kDa range, a 12-230 kDa separation system is recommended

• Exposure Time: Optimization may be required based on expression level in specific samples

How can researchers effectively use THBS4B antibodies for immunohistochemistry and immunofluorescence?

Effective immunohistochemical and immunofluorescence detection of THBS4B requires attention to several methodological details:

For Immunohistochemistry (Fixed Tissues):

• Fixation: Both paraffin-embedded and frozen sections have been successfully used

• Antigen Retrieval: May be necessary depending on fixation method (consult specific antibody documentation)

• Blocking: Thorough blocking is critical to reduce background, especially in vascular and inflammatory tissues

• THBS4B antibodies have been validated for detection in various tissues including vascular lesions in mouse models

For Immunofluorescence:

• For co-localization studies with macrophage markers, the following approach has proven effective:

  • Primary staining: Anti-THBS4 antibody and anti-CD68 (macrophage marker)

  • Secondary detection: Fluorophore-conjugated secondaries (red channel for THBS4, green channel for macrophages)

  • Nuclear counterstain: DAPI (blue)

• For cultured cells (e.g., RAW264.7 and BMDM):

  • LPS treatment (0.5 μg/ml for 24h) induces detectable THBS4 expression

  • Green fluorescence channel has been effectively used for THBS4 visualization with DAPI nuclear counterstain

Established Protocol Example:
LPS-induced THBS4 expression in macrophages has been visualized using the following approach:

• Treat cells with 0.5 μg/ml LPS for 24 hours

• Fix and permeabilize cells according to standard protocols

• Stain with anti-THBS4 antibody followed by appropriate secondary antibody

• Counterstain nuclei with DAPI

• Visualization shows green THBS4 signal in LPS-treated cells with minimal background in untreated controls

What methods are recommended for validating the specificity of THBS4B antibodies?

Antibody validation is critical for ensuring reliable research outcomes. For THBS4B antibodies, multiple complementary approaches are recommended:

Genetic Controls:

• Negative control validation using Thbs4−/− knockout tissues or cells is the gold standard

• Published studies have confirmed antibody specificity by demonstrating absence of staining in aortic lesions from Thbs4−/− mice

Cross-Species Specificity Testing:

• When studying zebrafish THBS4B, researchers have validated antibody specificity by confirming that human THBS4 antibodies do not cross-react with zebrafish THBS4 protein

• This specificity testing is particularly important in developmental studies where evolutionary conservation may vary

Molecular Weight Verification:

• Western blot confirmation of the expected molecular weight band (approximately 130-158 kDa for human THBS4)

• Simple Western™ analysis can provide additional verification with high sensitivity

Peptide Competition Assays:

• Pre-incubation of the antibody with the immunizing peptide should abolish specific staining

• This approach can be particularly valuable when knockout controls are unavailable

Expression Induction Studies:

• LPS treatment of macrophages (0.5 μg/ml for 1-24h) increases THBS4 expression in a time-dependent manner that can be detected by Western blotting and immunofluorescence, providing a positive control system

How does THBS4 function in stress response pathways, and what experimental approaches best capture these dynamics?

THBS4 plays a crucial role in cellular stress response pathways, particularly in the endoplasmic reticulum (ER) stress response. To effectively study these dynamics, researchers should consider the following experimental approaches:

ER Stress Response Analysis:
THBS4 directly interacts with the ER stress response machinery by binding the ER luminal domain of ATF6α, facilitating its translocation to the Golgi and subsequently to the nucleus . This activates genes associated with adaptive ER stress response. To capture this process:

• Co-immunoprecipitation assays can detect THBS4-ATF6α interaction

• Subcellular fractionation combined with Western blotting can track ATF6α translocation

• Reporter assays using ATF6-responsive elements can quantify pathway activation

Protein Synthesis and Degradation Studies:
THBS4's role in stress response can be investigated using cycloheximide (CHX) chase assays. Published protocols have utilized:

• CHX (25 μM) treatment for 1-5 hours

• Combined CHX (25 μM) + LPS (0.5 μg/mL) treatment for 1-3 hours

• Western blot detection of THBS4 with β-actin as loading control

Stress-Induced Expression Changes:
LPS-induced stress in macrophages provides a model system for studying THBS4 dynamics:

• qRT-PCR analysis reveals significant upregulation following LPS treatment

• Sequential time points (1-24h) demonstrate the temporal dynamics of the response

What role does THBS4B play in vascular inflammation, and how can researchers investigate this function?

THBS4 has been implicated in regulating vascular inflammation, with significant effects on atherosclerotic lesion development. Researchers investigating this function should consider:

Atherosclerotic Lesion Analysis:
Studies comparing ApoE−/− mice with Thbs4−/−/ApoE−/− double knockout mice have revealed important insights into THBS4's role in lesion development . Key experimental approaches include:

• Quantitative histological analysis of lesion cellular composition:

  • Measure area occupied by nuclei (hematoxylin staining)

  • Include fibrous cap and endothelial cells in analysis

  • Compare wild-type versus knockout models systematically

• Macrophage accumulation assessment:

  • THBS4 promotes macrophage accumulation in peritoneal tissue during inflammation

  • Quantitative analysis of macrophage numbers in peritoneal cavity provides a measurable endpoint

  • Statistical analysis (n=5 per group) has demonstrated significant differences (p<0.05)

• Expression analysis in inflammatory conditions:

  • qRT-PCR analysis of THBS4 expression in macrophages from peritoneal cavity lavage

  • Measurement of fold increase (RQ) over control values

  • Correlation with inflammatory markers

Co-localization Studies:
Immunofluorescence approaches combining macrophage markers (CD68) with THBS4 staining in peritoneal tissue provide powerful visualization of THBS4's role in inflammation:

• Blue = nuclei (DAPI)

• Green = macrophages (anti-CD68)

• Red = THBS4 (anti-TSP-4)

What are the key technical considerations when studying THBS4B in developmental biology and zebrafish models?

Zebrafish models offer valuable insights into THBS4B function during development. Researchers working with these models should consider several technical aspects:

Antibody Selection and Validation:

• Rabbit Polyclonal TSP4B antibody targeting the N-terminal region (aa 1-350) of zebrafish THBS4B has been successfully used

• Cross-reactivity testing is essential - human THBS4 antibodies do not cross-react with zebrafish THBS4 protein, allowing for specific detection of injected human protein versus endogenous zebrafish protein

Experimental Approaches:

• Microinjection of human TSP4 protein into zebrafish embryos followed by immunodetection has been published:

  • Detection of human TSP4 protein in the red channel

  • Confirmation of antibody specificity by lack of cross-reaction with zebrafish Tsp4 protein

  • Published results in eLife (2014;3:e02372)

Developmental Context:

• THBS4 regulates skeletal muscle integrity and susceptibility to muscular dystrophy through membrane organization

• When studying developmental phenotypes, careful staging and documentation of embryonic development is essential

• Comparative analysis between wild-type and THBS4-deficient models can reveal stage-specific requirements

What are common challenges in THBS4B detection, and how can researchers overcome them?

Researchers working with THBS4B antibodies may encounter several technical challenges. Here are evidence-based solutions to common problems:

Inconsistent or Weak Western Blot Signals:

• Problem: Variable or weak bands when detecting THBS4

• Solutions:

  • Optimize protein loading: 0.2 mg/mL of heart tissue lysate has been demonstrated to provide optimal results

  • Adjust antibody concentration: For human THBS4, 0.2-2 μg/mL of antibody has proven effective

  • Consider separation system: A 12-230 kDa separation system is recommended for the 130-158 kDa THBS4 protein

  • Use freshly prepared samples, as THBS4 may be subject to degradation

Background in Immunohistochemistry/Immunofluorescence:

• Problem: High background or non-specific staining

• Solutions:

  • Include proper negative controls: Thbs4−/− tissues have confirmed the specificity of staining in published studies

  • Optimize blocking: Extended blocking with appropriate serum can reduce background

  • Titrate antibody concentration: Start with manufacturer recommendations and adjust as needed

  • Consider tissue-specific autofluorescence: This can be particularly important in vascular and cardiac tissues

Species Cross-Reactivity Issues:

• Problem: Uncertainty about antibody specificity across species

• Solutions:

  • Validate species specificity experimentally: Human TSP4 protein injection into zebrafish embryos demonstrated that anti-human THBS4 antibody did not cross-react with zebrafish Tsp4 protein

  • Review published validation data: Many antibodies have been tested in multiple species with results documented in publications

How should researchers design experiments to differentiate between the intracellular and extracellular functions of THBS4B?

THBS4 has both traditional extracellular matrix functions and newly appreciated intracellular roles, particularly in ER stress response. Differentiating between these functions requires careful experimental design:

Subcellular Fractionation Approaches:

• Sequential extraction protocols can separate cytosolic, membrane, nuclear, and ECM-associated THBS4

• Western blot analysis of fractions with appropriate compartment markers (e.g., BiP/GRP78 for ER, α-tubulin for cytosol)

• This approach has helped establish THBS4's presence within the ER lumen

Functional Assays for Extracellular Roles:

• Cell-to-cell and cell-to-matrix adhesion assays with THBS4-coated substrates

• Analysis of cellular proliferation, migration, and attachment in the presence of exogenous THBS4

• Blocking antibodies applied extracellularly can help determine the contribution of secreted THBS4

ER Stress Response Pathway Analysis:

• ATF6α activation and nuclear translocation studies

• Detection of ATF6α target gene expression via qRT-PCR following THBS4 manipulation

• These approaches have established THBS4's role in mediating adaptive ER stress response

Genetic Manipulation Strategies:

• Expression of THBS4 with modified targeting sequences to direct localization

• Comparison of secreted versus ER-retained THBS4 constructs

• Phenotypic rescue experiments in Thbs4−/− models with compartment-specific expression

What analytical methods are recommended for quantifying THBS4B expression across different experimental systems?

Accurate quantification of THBS4B expression is critical for comparative studies. The following analytical methods have been validated in published research:

Western Blot Quantification:

• Densitometric analysis with appropriate loading controls (β-actin has been successfully used)

• Simple Western™ analysis provides automated quantification with high reproducibility

• Expected molecular weight ranges: 130 kDa (reducing conditions) to 158 kDa (Simple Western™ system)

qRT-PCR for Transcriptional Analysis:

• Successfully used to quantify THBS4 expression in:

  • Macrophages from peritoneal cavity lavage

  • Peritoneal tissue from control and LPS-treated mice

  • Cultured RAW264.7 cells and bone marrow-derived macrophages

• Results typically reported as fold increase (RQ) over control values

• Statistical significance established with p < 0.05, n = 3

Immunohistochemical Quantification:

• Area measurement of lesions occupied by nuclei (hematoxylin staining)

• Inclusion of fibrous cap and endothelial cells in analysis

• Quantification of cell numbers and distribution patterns in lesions

ELISA and Luminex Analysis:

• Sandwich ELISA using monoclonal antibody (MAB2390) in combination with polyclonal antibody (AF2390) and recombinant protein (2390-TH) has been validated for detecting TSP4 in human serum and plasma

• Luminex platform has also been applied successfully for THBS4 quantification in serum samples

What emerging applications of THBS4B antibodies should researchers consider exploring?

As our understanding of THBS4 biology expands, several promising research directions emerge:

Single-Cell Analysis Applications:

• Integration of THBS4B antibodies into single-cell proteomics workflows

• Investigation of cell-specific expression patterns in heterogeneous tissues like atherosclerotic plaques

• Correlation of THBS4 expression with cell state and phenotypic markers

Therapeutic Targeting Approaches:

• Development of function-blocking antibodies targeting specific THBS4 domains

• Investigation of THBS4-mediated ER stress pathways as potential intervention points

• Assessment of THBS4 as a biomarker for vascular inflammation or muscular dystrophy progression

Advanced Imaging Applications:

• Super-resolution microscopy to visualize THBS4 interactions with cellular components

• Live-cell imaging with tagged THBS4 antibody fragments to track dynamics

• Multiparametric imaging combining THBS4 with other markers of cellular stress and inflammation

How might THBS4B function across different disease models, and what methodological approaches are needed?

THBS4 has been implicated in multiple disease contexts, each requiring specific methodological considerations:

Cardiovascular Disease Models:

• ApoE−/−/Thbs4−/− double knockout mice have revealed THBS4's role in atherosclerotic lesion development

• Key methodologies include:

  • Quantitative histopathology of lesion composition

  • Analysis of inflammatory cell infiltration

  • Correlation with vascular function parameters

Muscular Dystrophy Research:

• THBS4 regulates skeletal muscle integrity and susceptibility to muscular dystrophy

• Methodological approaches include:

  • Muscle function testing in wild-type versus THBS4-deficient models

  • Analysis of membrane organization and stability

  • Assessment of ER stress response pathways in muscle tissue

Inflammatory Disease Models:

• LPS-induced peritonitis models have demonstrated THBS4's role in macrophage accumulation

• Key methodologies include:

  • Quantification of inflammatory cell recruitment

  • Assessment of cytokine/chemokine profiles

  • Correlation with tissue pathology

Developmental Disorders:

• Zebrafish models have provided insights into THBS4B's developmental functions

• Methodological considerations include:

  • Temporal analysis of expression during development

  • Phenotypic characterization of THBS4B-deficient embryos

  • Rescue experiments with domain-specific constructs