STAMBP Antibody

What is STAMBP and why is it a relevant research target?

STAMBP (STAM-binding protein), also known as AMSH, is a JAMM metalloprotease in the deubiquitinase (DUB) family. It functions primarily as an endosome-associated DUB by interacting with the SH3 domain of STAM, a component in the endosomal sorting complexes required for transport (ESCRT) machinery . STAMBP plays critical roles in:

• Regulating endosomal sorting and recycling

• Maintaining cellular homeostasis

• Modulating inflammasome activation

• Cancer progression in several malignancies

Research significance has increased as STAMBP dysregulation has been linked to various diseases, including cancer and neurodegenerative disorders . Notably, elevated STAMBP expression correlates with poor clinical prognosis in multiple cancers, such as lung adenocarcinoma, breast cancer, triple-negative breast cancer, and pancreatic cancer .

What are the standard applications for STAMBP antibodies in research?

Based on validated antibodies in the search results, STAMBP antibodies can be reliably used for:

When selecting an antibody, researchers should consider the specific reactivity (human, mouse, rat) and whether the experimental design requires polyclonal or monoclonal antibodies .

How can researchers validate STAMBP antibody specificity?

For proper validation of STAMBP antibody specificity, implement the following methodological approach:

• Positive controls: Use cell lines known to express STAMBP, such as HT-29 human colon adenocarcinoma cells, which have been validated to express detectable levels of STAMBP by western blot .

• Knockdown validation: Compare antibody reactivity between normal cells and those with STAMBP knockdown via siRNA or CRISPR/Cas9. The search results show that shSTAMBP plasmid effectively decreased STAMBP expression as validated by western blotting and qRT-PCR analyses .

• Cross-reactivity testing: Verify that STAMBP antibodies don't cross-react with other JAMM deubiquitinase family members. Western blot analysis has confirmed that altering STAMBP does not affect other proteins in the Zn+-dependent JAMM deubiquitinases family .

• Multiple detection methods: Confirm findings using more than one technique (e.g., WB, IHC, and IF) to ensure consistent results across platforms .

How does STAMBP contribute to cancer chemoresistance mechanisms?

STAMBP plays a significant role in chemoresistance, particularly in pancreatic cancer through the STAMBP-E2F1-PDK1 axis:

• PDK1-mediated metabolic reprogramming: STAMBP enhances aerobic glycolysis and suppresses mitochondrial respiration to increase gemcitabine resistance in pancreatic cancer cells. This occurs through stabilization of PDK1, an essential regulator of the aerobic glycolytic process .

• E2F1 stabilization mechanism: STAMBP promotes PDK1-mediated Warburg effect and chemotherapy resistance by modulating E2F1. It directly binds to E2F1 and suppresses its degradation and ubiquitination .

• Experimental evidence: Knockdown of STAMBP significantly increased gemcitabine sensitivity in pancreatic cancer cells both in vitro and in vivo. In TCGA cohort analysis, patients with low STAMBP expression showed better response to gemcitabine treatment, while approximately 80.6% of high STAMBP-expressing patients exhibited progressive disease after treatment .

• Therapeutic potential: Entrectinib, an FDA-approved drug, was identified as a STAMBP inhibitor that enhances gemcitabine effectiveness in a patient-derived xenograft model, suggesting a potential approach to overcome chemoresistance .

What is the role of STAMBP in regulating inflammasome activity?

STAMBP modulates inflammasome activation and cytokine secretion through multiple mechanisms:

• NLRP3 deubiquitination: STAMBP regulates NLRP3 inflammasome activity by controlling its K63-linked polyubiquitination. In monocytes, STAMBP knockout increased NLRP3 K63 chain polyubiquitination resulting in increased NLRP3 inflammasome activation .

• NALP7 protein stabilization: STAMBP stabilizes NALP7 by preventing its lysosomal degradation through deubiquitination. Using a cell-free DUB assay, researchers demonstrated that recombinant STAMBP directly deubiquitinates NALP7 in a time-dependent manner .

• IL-1β regulation: STAMBP knockout in monocytes increased expression of numerous cytokines and chemokines in response to TLR agonists or LPS. This exaggerated inflammatory response was dependent on IL-1β signaling, as STAMBP knockout directly increased IL-1β release with TLR ligation .

• Pharmacological targeting: Small-molecule inhibitor BC-1471 decreases NALP7 protein levels and suppresses IL-1β release after TLR agonism, suggesting potential therapeutic applications for inflammatory conditions .

What experimental approaches are effective for studying STAMBP-protein interactions?

For investigating STAMBP interactions with partner proteins, researchers should consider:

• Co-Immunoprecipitation: Use anti-STAMBP antibodies (0.5μg-4μg) with 200-400μg extracts of whole cells for IP experiments . This approach was successfully used to demonstrate STAMBP's interaction with E2F1 in pancreatic cancer research .

• Immunoprecipitation-Mass Spectrometry: This combined approach identified the interaction between STAMBP and the actin-binding protein RAI14 in triple-negative breast cancer cells .

• Cell-free deubiquitination assays: Develop assays using recombinant STAMBP and immunopurified ubiquitinated substrates to directly test STAMBP's deubiquitination activity, as demonstrated with Ub-NALP7 .

• Proximity ligation assays: For detecting in situ protein-protein interactions within cells to confirm STAMBP binding partners identified through other methods.

• Functional validation: Confirm the significance of identified interactions through genetic manipulation (knockdown/overexpression) of STAMBP and assessment of target protein stability, as demonstrated with RAI14 in TNBC .

What are the optimal sample preparation methods for STAMBP detection?

To ensure reliable STAMBP detection in different experimental contexts:

• For Western blotting:

  • Use PVDF membrane for optimal protein transfer

  • Run under reducing conditions with appropriate immunoblot buffer groups

  • A specific band for STAMBP should be detected at approximately 50 kDa

  • Include protease inhibitors in lysis buffers to prevent degradation

• For Immunohistochemistry:

  • Use immersion-fixed paraffin-embedded sections

  • Apply antigen retrieval techniques if necessary

  • STAMBP typically shows cytoplasmic localization in epithelial cells

  • A MaxVision kit has been successfully used for STAMBP immunostaining in tumor xenograft sections

• For Immunofluorescence/ICC:

  • Optimize fixation method (paraformaldehyde is commonly used)

  • Include permeabilization step to access intracellular STAMBP

  • Use appropriate blocking solutions to minimize background

How can researchers effectively manipulate STAMBP expression in experimental models?

The search results demonstrate several validated approaches:

• RNA interference:

  • siRNA has been successfully used to silence STAMBP expression in THP-1 cells, resulting in decreased NALP7 protein abundance

  • Validated shSTAMBP plasmids have effectively decreased STAMBP expression as confirmed by western blotting and qRT-PCR analyses

• CRISPR/Cas9 gene editing:

  • Single clones of THP-1 STAMBP knockout monocytes were generated via lentiviral transduction using the lentCRISPRv2 backbone with targeting sgRNA and selection with puromycin

  • This approach created stable STAMBP-/- and Cas9 control THP-1 cell lines for studying inflammasome regulation

• Overexpression systems:

  • Lentiviral infection with plasmids encoding STAMBP has been used to rescue STAMBP levels in knockdown models

  • Ectopic expression of STAMBP in HeLa and Beas2B cells successfully increased NALP7 protein levels

• In vivo models:

  • Nude mice injected with stably knocked-down STAMBP-PC cells treated with gemcitabine showed significantly decreased tumor volume compared to control groups

  • Patient-derived xenograft (PDX) models have been used to test STAMBP inhibitors in combination with chemotherapy

What controls should be included when studying STAMBP's role in disease mechanisms?

Based on published research methodologies, include these critical controls:

• For gene expression studies:

  • Compare STAMBP expression in tumor tissues versus adjacent normal tissues

  • Include survival analysis correlating STAMBP expression with patient outcomes

  • Stratify patients by STAMBP expression levels (high/low based on median values)

• For protein stability experiments:

  • Include cycloheximide (CHX) chase assays to measure protein decay rates with and without STAMBP manipulation

  • Compare multiple timepoints to establish degradation kinetics

• For ubiquitination studies:

  • Examine both K48-linked (degradative) and K63-linked (non-degradative) ubiquitination patterns

  • Include proteasome inhibitors (e.g., MG132) to prevent degradation of ubiquitinated proteins

  • Verify specificity by examining multiple ubiquitination targets

• For in vivo studies:

  • Include both vehicle control and treatment groups

  • Measure multiple parameters including tumor volume, weight, and survival period

  • Validate findings with Ki67 immunohistochemistry to confirm effects on cell proliferation

How can researchers address inconsistent results in STAMBP detection experiments?

When encountering inconsistent results with STAMBP antibodies:

• Antibody validation:

  • Verify antibody specificity using positive controls (e.g., HT-29 human colon adenocarcinoma cell line)

  • Test multiple antibodies targeting different epitopes of STAMBP

  • Consider the immunogen used to generate the antibody - some use recombinant fusion protein containing sequences corresponding to amino acids 100-270 of human STAMBP

• Sample preparation issues:

  • Ensure complete protein denaturation for western blotting

  • Optimize fixation protocols for immunohistochemistry and immunofluorescence

  • Include appropriate protease inhibitors in lysis buffers

• Technical considerations:

  • Adjust antibody dilution within recommended ranges (e.g., 1:500-1:2000 for WB)

  • Optimize incubation times and temperatures

  • Use fresh antibodies and avoid repeated freeze-thaw cycles which can compromise activity

• Cell type variations:

  • STAMBP expression levels vary across cell types

  • Consider that STAMBP protein levels may increase after treatments like gemcitabine

What are the best approaches for analyzing STAMBP's role in complex disease mechanisms?

To comprehensively analyze STAMBP's role in diseases like cancer:

• Multi-omics integration:

  • Combine transcriptomics, proteomics, and metabolomics analyses as demonstrated in pancreatic cancer studies

  • Use bioinformatics tools to analyze clinical tissue sample databases for STAMBP expression patterns

• Pathway analysis:

  • Investigate STAMBP-E2F1-PDK1 axis in cancer chemoresistance

  • Examine STAMBP's impact on aerobic glycolysis versus mitochondrial respiration

  • Analyze STAMBP's role in RAI14 stabilization in triple-negative breast cancer

• Clinical correlation:

  • Evaluate STAMBP expression in patient samples and correlate with clinical outcomes

  • Stratify patients by STAMBP expression levels to identify potential biomarker applications

  • Assess connection between STAMBP expression and treatment response

• Drug sensitivity testing:

  • Screen compound libraries for STAMBP inhibitors, as demonstrated with entrectinib

  • Test combinations of STAMBP inhibitors with standard chemotherapeutics

  • Use three-dimensional protein structure analysis to identify potential binding sites for STAMBP inhibitors

How can researchers distinguish between STAMBP's direct and indirect effects in cellular pathways?

To differentiate between direct and indirect STAMBP effects:

• In vitro deubiquitination assays:

  • Use purified recombinant STAMBP with immunopurified ubiquitinated substrates to demonstrate direct deubiquitination

  • Include time-dependent analysis to establish enzymatic kinetics

• Rescue experiments:

  • Restore wild-type STAMBP in knockout models to confirm phenotype reversibility

  • Use catalytically inactive STAMBP mutants to determine if enzymatic activity is required for observed effects

• Domain-specific mutations:

  • Generate STAMBP constructs with mutations in specific functional domains

  • Test which domains are essential for specific protein interactions or cellular functions

• Temporal analysis:

  • Use inducible expression/knockdown systems to track immediate versus delayed effects of STAMBP manipulation

  • Combine with time-course analyses of downstream events to establish causality

• Direct binding confirmation:

  • Perform structural studies or domain mapping to confirm direct binding between STAMBP and partner proteins, as demonstrated with E2F1

  • Use proximity ligation assays to verify protein-protein interactions in intact cells

By implementing these methodological approaches, researchers can more confidently distinguish STAMBP's direct enzymatic and binding functions from secondary pathway effects.

How does STAMBP expression correlate with clinical outcomes in cancer patients?

Multiple studies have established significant correlations between STAMBP expression and clinical outcomes:

These clinical correlations suggest STAMBP could serve as both a prognostic biomarker and potential therapeutic target in multiple cancer types.

What methodologies are effective for evaluating STAMBP as a therapeutic target?

To evaluate STAMBP as a potential therapeutic target:

• High-throughput screening approaches:

  • Compound library screening using three-dimensional protein structure analysis identified entrectinib as a STAMBP inhibitor

  • Develop cell-based assays to screen for compounds that decrease STAMBP activity or expression

• Preclinical model testing:

  • Patient-derived xenograft (PDX) models have been used to test STAMBP inhibitors in combination with chemotherapy

  • Compare tumor growth, invasion, and metastasis in models with and without STAMBP inhibition

• Combination therapy assessment:

  • Test STAMBP inhibitors in combination with standard chemotherapeutics

  • Evaluate whether STAMBP inhibition can overcome established chemoresistance, as demonstrated with entrectinib and gemcitabine in pancreatic cancer

• Biomarker development:

  • Correlate STAMBP expression levels with treatment response

  • Develop standardized assays to measure STAMBP expression in clinical samples

  • Evaluate whether STAMBP inhibition affects downstream biomarkers like PDK1 or E2F1 expression