SERPINB5 Antibody

What is SERPINB5 and how is it classified molecularly?

SERPINB5 (Serpin B5) belongs to the serpin (serine protease inhibitor) superfamily. Despite this classification, it is considered a non-classical serpin that does not exhibit typical serine protease inhibitory activity as it does not undergo the characteristic S (stressed) to R (relaxed) conformational transition. In humans, SERPINB5 is encoded by the SERPINB5 gene and functions primarily as a tumor suppressor in epithelial cells. It has a calculated molecular weight of approximately 42 kDa and plays crucial roles in suppressing cancer cell invasion and metastasis while also functioning as an angiogenesis inhibitor .

Where is SERPINB5 predominantly expressed in normal tissues?

SERPINB5 expression exhibits a tissue-specific pattern, being predominantly found in the skin, prostate, testis, intestine, tongue, lung, and thymus. This expression pattern is significant for researchers designing tissue-specific studies or selecting appropriate controls. The protein's cellular localization has been characterized as secreted in the extracellular space, which informs experimental approaches for detection and functional studies .

What applications are SERPINB5 antibodies validated for?

Commercial SERPINB5 antibodies have been validated for multiple experimental applications, primarily Western blotting (WB) and immunohistochemistry (IHC). When selecting an antibody for Western blotting, recommended dilution ranges typically fall between 1:1000-1:5000, while IHC applications generally require 1:100-1:300 dilutions. Verification studies have demonstrated successful detection in specific tissue samples including mouse esophagus, human cervical cancer, human esophagus cancer, and human lung cancer .

How does SERPINB5 modulate cell-cell adhesion mechanisms?

Recent research has revealed that SERPINB5 plays a crucial role in maintaining intercellular adhesion by interacting with and regulating the localization of desmoplakin (DSP) at the plasma membrane. Mechanistically, SERPINB5 functions through modulation of TGF-β signaling, which is known to destabilize DSP in keratinocytes. Overexpression of SERPINB5 has been shown to prevent autoantibody-mediated loss of cell-cell adhesion by stabilizing DSP at the membrane, though notably it does not affect DSG3 (desmoglein 3) membrane levels. This selective regulation suggests a specialized function in maintaining desmosomal integrity .

What is the relationship between SERPINB5 and TGF-β signaling?

SERPINB5 exhibits a complex regulatory relationship with TGF-β signaling pathways. Knockdown experiments demonstrate that SERPINB5 depletion leads to significantly enhanced levels of phosphorylated SMAD2/3 and SMAD1/3/5, key downstream effectors of TGF-β signaling. This indicates SERPINB5 functions as a negative modulator of this pathway. Conversely, TGF-β signaling can positively modulate SERPINB5, suggesting the existence of a feedback loop. This relationship has significant implications for intercellular adhesion, as TGF-β signaling negatively regulates DSP localization at the cell membrane. Inhibition of TGF-β activation with small molecule inhibitors such as GW788388 can rescue the loss of cell-cell adhesion associated with SERPINB5 knockdown .

What controls should be included when working with SERPINB5 antibodies?

When designing experiments with SERPINB5 antibodies, researchers should include:

• Positive tissue/cell controls: Based on validated samples, mouse esophagus, human cervical cancer, human esophagus cancer, and human lung cancer tissues serve as reliable positive controls. Cell lines including HeLa (cervical epithelial carcinoma), PC-3 (prostate cancer), and RT-4 (bladder carcinoma) have demonstrated detectable SERPINB5 expression .

• Negative controls: Include samples known to lack SERPINB5 expression or use isotype control antibodies to confirm specificity.

• Loading/staining controls: Standard protein loading controls for Western blots and appropriate staining controls for IHC applications to ensure technical consistency.

• Knockdown/knockout validation: Where possible, SERPINB5-depleted samples provide valuable specificity controls.

How should SERPINB5 antibodies be stored and handled?

For optimal antibody performance, SERPINB5 antibodies should be stored at -20°C, where they typically remain stable for up to 12 months. After reconstitution, the antibody can be stored at 4°C for up to one month, but for longer-term storage, aliquoting and maintaining at -20°C is recommended. Repeated freeze-thaw cycles should be avoided as they can compromise antibody activity and specificity. During shipping and short-term handling, cold packs are typically used to maintain antibody integrity .

What cell lines are most suitable for SERPINB5 expression studies?

Based on detection validation studies, several cell lines have demonstrated reliable SERPINB5 expression and are suitable for expression studies:

• HeLa human cervical epithelial carcinoma cells

• PC-3 human prostate cancer cells

• RT-4 human bladder carcinoma cells

These cell lines provide consistent expression profiles and can serve as experimental models for investigating SERPINB5 function in various cellular contexts .

What methodological approaches are effective for studying SERPINB5's role in autoimmune blistering disorders?

For investigating SERPINB5's role in autoimmune blistering disorders such as pemphigus vulgaris (PV), a multi-faceted methodological approach is recommended:

• Dispase-based cell cohesion assays: These assays can quantitatively measure changes in intercellular adhesion strength following manipulation of SERPINB5 expression and/or exposure to pathogenic autoantibodies.

• Immunofluorescence microscopy: This technique allows visualization of DSP and DSG3 localization patterns at the cell membrane, with particular attention to changes in distribution patterns following SERPINB5 overexpression or knockdown.

• Ex-vivo skin models: Human ex-vivo skin models treated with PV autoantibodies provide a physiologically relevant system to evaluate blister formation and the potential protective effects of manipulating SERPINB5 expression or inhibiting TGF-β signaling.

• Patient sample analysis: Examination of skin biopsies from PV patients compared to healthy controls, focusing on SERPINB5 expression levels, TGF-β pathway activation (pSMAD2/3 levels), and desmosomal protein localization .

How can researchers effectively investigate SERPINB5-TGF-β signaling interactions?

To comprehensively investigate the interactions between SERPINB5 and TGF-β signaling, researchers should consider the following methodological approach:

• Phosphorylation state analysis: Western blotting with phospho-specific antibodies against SMAD2/3 and SMAD1/3/5 to assess TGF-β pathway activation.

• Genetic manipulation studies: SERPINB5 knockdown and overexpression experiments to observe corresponding changes in TGF-β pathway activation markers.

• Pharmacological intervention: Use of TGF-β pathway inhibitors (e.g., GW788388) to determine if they can rescue phenotypes associated with SERPINB5 depletion.

• Protein-protein interaction studies: Co-immunoprecipitation or proximity ligation assays to identify direct interactions between SERPINB5 and components of the TGF-β signaling pathway.

• Reporter gene assays: SMAD-responsive luciferase reporters to quantitatively measure TGF-β pathway activity in response to SERPINB5 modulation .

How is SERPINB5 expression altered in cancer tissues?

SERPINB5 was first identified as a tumor suppressor in breast cancer and has since been associated with altered expression in multiple cancer types. Its tumor suppressor function manifests through inhibition of migration, invasion, and metastatic properties, particularly in mammary tumors. SERPINB5 antibodies have been successfully used to detect expression in cervical cancer, esophageal cancer, lung cancer, prostate cancer, and bladder carcinoma samples. The dysregulation of SERPINB5 expression in these cancers provides important insights into potential diagnostic and therapeutic applications .

What is the significance of SERPINB5 in cell adhesion disorders?

SERPINB5 has emerged as a significant regulator of intercellular adhesion, particularly in the context of desmosomal adhesion. Recent research has demonstrated that SERPINB5 plays a protective role in autoimmune skin blistering disorders such as pemphigus vulgaris (PV), where impairment of desmosomal cell-cell adhesion leads to life-threatening disease. SERPINB5 overexpression has been shown to prevent PV-IgG-mediated loss of cell-cell adhesion and the displacement of desmoplakin from the cell membrane, highlighting its potential as a therapeutic target for stabilizing intercellular adhesion in disease states .

How might targeting SERPINB5-regulated pathways offer therapeutic potential in pemphigus vulgaris?

The identification of SERPINB5 as a modulator of desmoplakin membrane localization through TGF-β signaling opens novel therapeutic avenues for pemphigus vulgaris treatment:

• TGF-β pathway inhibition: Elevated TGF-β activation observed in PV patient samples suggests that inhibitors of this pathway may serve as a targeted therapeutic option. Experimental evidence demonstrates that inhibition of TGF-β activation ameliorates PV-IgG-mediated loss of cell-cell adhesion, increases DSP membrane expression, and prevents PV-IgG-induced blister formation in human ex-vivo skin models.

• SERPINB5 enhancement strategies: Approaches that increase SERPINB5 expression or activity could potentially stabilize desmosomal adhesion in PV patients.

• Combination therapies: Integrating TGF-β inhibition with existing immunosuppressive treatments may provide more comprehensive disease management by addressing both autoimmune responses and adhesion defects.

• Topical application considerations: Given the skin-specific nature of PV manifestations, development of topical formulations of TGF-β inhibitors might provide targeted treatment with reduced systemic side effects .

What methodological challenges exist in studying SERPINB5's functional mechanisms in disease contexts?

Researchers face several methodological challenges when investigating SERPINB5's roles in disease:

• Tissue and context specificity: SERPINB5 functions appear to be highly tissue-specific and context-dependent, requiring careful selection of experimental models that accurately recapitulate the physiological environment.

• Pathway crosstalk complexity: SERPINB5 interacts with multiple signaling pathways, including TGF-β and potentially others, creating complex regulatory networks that are challenging to dissect.

• Translation to in vivo contexts: While in vitro and ex vivo models provide valuable insights, translating findings to complete in vivo systems presents technical hurdles.

• Temporal dynamics: SERPINB5's involvement in feedback regulation with TGF-β signaling introduces temporal complexities that require sophisticated experimental designs to fully characterize.

• Disease heterogeneity: Individual patient variations in disease mechanisms and SERPINB5 expression patterns necessitate personalized approaches to fully understand its roles in pathogenesis .

What factors can affect SERPINB5 antibody performance in Western blotting?

Several factors can influence SERPINB5 antibody performance in Western blotting applications:

• Sample preparation: Complete protein denaturation is essential; use of appropriate buffers containing 50% glycerol and pH stabilization at 7.4 helps maintain antibody recognition of the target epitope.

• Dilution optimization: While recommended ranges are 1:1000-1:5000, optimal dilution should be determined empirically for each experimental system.

• Blocking conditions: Optimization of blocking reagents and times can reduce background and improve signal-to-noise ratio.

• Detection system compatibility: Ensure secondary antibody and detection systems are optimized for the host species (typically rabbit for polyclonal SERPINB5 antibodies).

• Protein loading: Excessive protein loading can lead to high background while insufficient loading may result in undetectable signals .

What are the optimal conditions for immunohistochemical detection of SERPINB5?

For effective immunohistochemical detection of SERPINB5:

• Antibody dilution: Start with the recommended range of 1:100-1:300 and optimize based on specific tissue type and fixation method.

• Antigen retrieval: This step is often critical for formalin-fixed, paraffin-embedded tissues to expose epitopes masked during fixation.

• Tissue selection: Human esophagus cancer and human lung cancer tissues have been verified as reliable positive controls.

• Incubation conditions: Optimize temperature and duration for primary antibody incubation to balance specific binding and background.

• Detection system: Choose an appropriate detection system compatible with the host species of the primary antibody (typically rabbit for polyclonal antibodies) .

What strategies can resolve inconsistent results when studying SERPINB5-TGF-β pathway interactions?

When encountering inconsistent results in SERPINB5-TGF-β pathway studies, consider these troubleshooting approaches:

• Baseline TGF-β activity assessment: Measure endogenous TGF-β signaling levels in your experimental system, as high baseline activity may mask effects of SERPINB5 manipulation.

• Temporal dynamics analysis: Implement time-course experiments to capture the dynamic nature of SERPINB5-TGF-β interactions, as signaling responses may be transient.

• Multiple readout parameters: Assess pathway activation using multiple markers beyond pSMAD2/3, including transcriptional targets of TGF-β signaling.

• Cell density standardization: Maintain consistent cell density across experiments, as confluence levels can significantly affect both SERPINB5 expression and TGF-β signaling.

• Validation across multiple cell types: Confirm findings in multiple epithelial cell types, as the relationship between SERPINB5 and TGF-β may vary between tissues.

• Combination of genetic and pharmacological approaches: Use both SERPINB5 knockdown/overexpression and TGF-β pathway inhibitors to comprehensively characterize the relationship .

How can researchers optimize co-localization studies of SERPINB5 with desmosomal proteins?

For effective co-localization studies of SERPINB5 with desmosomal proteins such as desmoplakin:

• Sequential antibody application: To minimize cross-reactivity, apply primary antibodies sequentially rather than simultaneously, particularly when antibodies are raised in the same host species.

• Super-resolution microscopy: Techniques such as STED, STORM, or PALM provide superior resolution compared to conventional confocal microscopy, allowing more precise determination of spatial relationships between SERPINB5 and desmosomal components.

• Z-stack acquisition: Collect images at multiple focal planes to create 3D reconstructions that more accurately represent the complex spatial organization of desmosomal structures.

• Quantitative co-localization analysis: Employ software tools that calculate Pearson's correlation coefficient or Manders' overlap coefficient to quantitatively assess the degree of co-localization.

• Live-cell imaging: Where possible, utilize tagged proteins for live-cell imaging to observe dynamic interactions between SERPINB5 and desmosomal components under various experimental conditions.

• Proximity ligation assays: This technique can detect protein interactions within 40 nm, providing stronger evidence for direct interactions between SERPINB5 and desmosomal proteins .

What recent discoveries have expanded our understanding of SERPINB5 function?

Recent research has significantly broadened our understanding of SERPINB5 beyond its initially described tumor suppressor role:

• Cell adhesion regulation: SERPINB5 has been identified as a positive regulator of desmoplakin membrane localization, directly impacting intercellular adhesive strength in keratinocytes.

• TGF-β pathway modulation: SERPINB5 has been established as a negative regulator of TGF-β signaling, with knockdown experiments demonstrating enhanced phosphorylation of SMAD proteins.

• Autoimmune disease relevance: Studies have linked SERPINB5 to pemphigus vulgaris pathogenesis, where overexpression can rescue autoantibody-mediated loss of cell-cell adhesion.

• Membrane protein trafficking: Beyond simply affecting expression levels, SERPINB5 appears to regulate the membrane trafficking and localization of key desmosomal components .

How might multi-omics approaches advance SERPINB5 research in disease contexts?

Integrative multi-omics approaches offer powerful tools for comprehensively understanding SERPINB5's roles in disease:

• Transcriptomic-proteomic integration: Combining RNA-seq with proteomics can identify discrepancies between SERPINB5 mRNA and protein levels, revealing post-transcriptional regulatory mechanisms.

• Phosphoproteomics: This approach can map SERPINB5-dependent changes in phosphorylation patterns across multiple signaling pathways, providing a systems-level view of its regulatory impact.

• Interaction proteomics: Techniques such as BioID or APEX proximity labeling coupled with mass spectrometry can identify the complete SERPINB5 interactome across different cellular compartments.

• Single-cell multi-omics: These methods can reveal cell-type-specific functions of SERPINB5 within heterogeneous tissues and capture the spectrum of responses to SERPINB5 modulation.

• Spatial transcriptomics/proteomics: These emerging technologies can map SERPINB5 expression and activity in the spatial context of tissues, providing insights into its function in maintaining tissue architecture .

What computational approaches show promise for predicting SERPINB5 functional interactions?

Advanced computational methods offer new avenues for predicting and understanding SERPINB5 functional interactions:

• Structural modeling: Despite being classified as a serpin, SERPINB5 does not undergo typical serpin conformational changes. Molecular dynamics simulations and AlphaFold-based structural predictions can provide insights into its unique structural properties and interaction interfaces.

• Network analysis algorithms: These can identify potential pathway connections and predict how SERPINB5 integrates into broader signaling networks, particularly its intersection with TGF-β pathway components.

• Machine learning approaches: These can predict cell- and tissue-specific SERPINB5 functions by integrating diverse data types including expression patterns, chromatin accessibility, and protein-protein interactions.

• Systems biology modeling: Differential equation-based models can capture the dynamic feedback relationship between SERPINB5 and TGF-β signaling, predicting system behavior under various perturbations.

• Text mining and knowledge graph approaches: These methods can synthesize information across the fragmented literature on SERPINB5, identifying connections that may not be apparent through traditional literature review .