KBTBD7 Antibody

What is KBTBD7 and why is it significant in cancer research?

KBTBD7 is a protein first cloned in 2010 that functions as both a transcriptional activator and substrate adaptor during ubiquitination processes. Its significance in cancer research stems from its recently discovered role in non-small cell lung cancer (NSCLC), where it promotes tumor progression through enhanced ubiquitin-dependent degradation of the tumor suppressor PTEN. This degradation subsequently activates the EGFR/PI3K/AKT signaling pathway, driving cancer cell proliferation and invasion. KBTBD7 has been found to be highly expressed in NSCLC tissues compared to normal specimens, with expression positively correlating with histological type, P-TNM stage, lymph node metastasis, and tumor size, suggesting its potential as a therapeutic target .

What cellular processes does KBTBD7 influence beyond cancer?

Beyond its emerging role in cancer, KBTBD7 has been implicated in multiple physiological and pathological processes. Studies have shown KBTBD7 involvement in excessive inflammation following myocardial infarction, where microRNA-21 can directly target it to prevent cardiac dysfunction. Additionally, KBTBD7 plays roles in brain development and neurofibromin stability. As a substrate adaptor of CUL3, KBTBD7 mediates ubiquitin-proteasome degradation of proteins including T-lymphoma and metastasis gene 1 (TIAM1) and dopamine type 2 receptor (DRD2), affecting TIAM1-RAC1 signaling and dopamine agonist resistance in pituitary adenoma, respectively .

How should researchers select the appropriate KBTBD7 antibody for specific experimental applications?

When selecting a KBTBD7 antibody, researchers should consider several factors based on their experimental needs. For immunohistochemistry applications, as demonstrated in studies of NSCLC tissues, anti-KBTBD7 rabbit antibodies (such as NBP3-05059 from Novus) have been successfully used at 1:100 dilution . Different applications (Western blotting, immunofluorescence, immunoprecipitation) may require antibodies with specific validation for those techniques. Researchers should examine the antibody's specificity documentation, including Western blot data showing the expected molecular weight (~51 kDa), cross-reactivity profile, and validation in relevant cell lines like SK, A549, H1975, H1299, and HCC827 where KBTBD7 is well-expressed . For co-immunoprecipitation studies involving protein-protein interactions, such as KBTBD7-PTEN binding, antibodies verified for immunoprecipitation applications are essential.

What are the most effective validation protocols for KBTBD7 antibodies in experimental settings?

Effective validation of KBTBD7 antibodies should employ multiple complementary approaches. Primary validation should include Western blotting with positive controls (NSCLC cell lines with known KBTBD7 expression) and negative controls (cells with KBTBD7 knockdown). Immunofluorescence validation should confirm the expected cytoplasmic localization of KBTBD7, as observed in NSCLC and HBE cell lines . For immunohistochemistry applications, researchers should validate using paired tumor and normal tissues, with appropriate scoring systems similar to those used in published studies (staining intensity categorized from 0-3, with area coverage from 1-4, yielding final scores of 0-12) . Advanced validation might include using CRISPR/Cas9 KBTBD7 knockout cells as negative controls, or overexpression systems as positive controls. Antibody specificity can be further confirmed by peptide competition assays and mass spectrometry verification of immunoprecipitated proteins.

What are the optimal conditions for using KBTBD7 antibodies in immunohistochemistry of NSCLC samples?

For optimal immunohistochemical detection of KBTBD7 in NSCLC samples, researchers should follow a protocol similar to that used in successful studies. Tissue specimens should be sectioned at 4 μm thickness and heated at 70°C for 4-6 hours prior to staining. After xylene deparaffinization and gradient alcohol hydration, antigen retrieval should be performed using EDTA repair solution at high temperature for 20 minutes. Anti-KBTBD7 rabbit antibody (such as NBP3-05059, Novus) has been effectively used at 1:100 dilution . For visualization, commercial IHC kits (such as those from MaixinBio) following manufacturer's protocols are recommended. For scoring, a combined approach evaluating both staining intensity (0-3) and percent area stained (1-4 scale) should be implemented, with a final multiplied score >6 considered positive for KBTBD7 expression . This methodology has successfully distinguished KBTBD7 expression patterns between NSCLC and adjacent non-cancerous tissues.

How should researchers design KBTBD7 knockdown experiments to study its function in cancer cells?

When designing KBTBD7 knockdown experiments to study its function in cancer cells, researchers should consider using multiple approaches for gene silencing. For transient knockdown, multiple siRNA sequences targeting different regions of KBTBD7 mRNA should be tested to identify the most effective construct while controlling for off-target effects. For stable knockdown, shRNA or CRISPR/Cas9 systems can be employed. Functional validation of knockdown efficiency should include both RT-PCR to confirm mRNA reduction and Western blot to verify protein depletion . Appropriate cell lines for these experiments include A549 and H1299, which have been successfully used in previous KBTBD7 studies . Following knockdown confirmation, researchers should employ multiple functional assays to comprehensively evaluate phenotypic changes, including proliferation assays (CCK-8, colony formation), invasion assays (Transwell), and analysis of downstream protein expression changes (CCNE1, CDK4, P27, ZEB-1, Claudin-1, ROCK1, MMP-9, E-cadherin) by Western blot . Rescue experiments using PTEN knockdown can help confirm the specificity of KBTBD7's effects through the PTEN-EGFR/PI3K/AKT axis .

What techniques are most effective for investigating the interaction between KBTBD7 and PTEN proteins?

For investigating the KBTBD7-PTEN interaction, co-immunoprecipitation (Co-IP) has proven effective in confirming their direct protein-protein binding . A comprehensive experimental approach should begin with reciprocal Co-IP, where either KBTBD7 or PTEN antibodies are used for immunoprecipitation, followed by immunoblotting for the other protein. For more sensitive detection, researchers can use tagged versions of these proteins (HA, FLAG, or GFP) in overexpression systems. Proximity ligation assays (PLA) can provide additional visualization of the interaction in situ within cells. For confirmation of direct interaction and detailed binding domain mapping, purified recombinant protein fragments representing different domains of KBTBD7 (particularly the BTB domain and Kelch repeats) and PTEN can be used in pull-down assays. Advanced techniques like FRET (Fluorescence Resonance Energy Transfer) or BiFC (Bimolecular Fluorescence Complementation) can provide real-time visualization of the interaction in living cells, while hydrogen-deuterium exchange mass spectrometry can offer detailed information about the interaction interface.

How can researchers effectively study KBTBD7-mediated ubiquitination of PTEN?

To study KBTBD7-mediated ubiquitination of PTEN, researchers should employ a comprehensive approach beginning with in vivo ubiquitination assays. As demonstrated in published protocols, NSCLC cells with stable KBTBD7 knockdown should be transfected with HA-tagged ubiquitin plasmid and pre-treated with the proteasome inhibitor MG-132 (typically 10 μM for 12 hours) before cell collection . PTEN should be immunoprecipitated using anti-PTEN antibody (#9188, Cell Signaling Technology or equivalent), followed by immunoblotting with anti-HA antibody to detect ubiquitinated PTEN species . To determine the specific type of ubiquitin linkage involved (K48 vs. K63), researchers can use linkage-specific antibodies or mutant ubiquitin constructs with individual lysine mutations. For in vitro verification, purified components (recombinant KBTBD7, PTEN, E1, E2, and ubiquitin) can be used in a reconstituted ubiquitination system. Mass spectrometry analysis of immunoprecipitated PTEN can further identify the specific lysine residues targeted for ubiquitination. Cycloheximide chase assays comparing PTEN degradation rates in control versus KBTBD7-depleted cells will help determine the functional impact of this ubiquitination on PTEN stability.

What experimental approaches can reveal how KBTBD7 affects the EGFR/PI3K/AKT pathway?

To comprehensively investigate KBTBD7's impact on the EGFR/PI3K/AKT pathway, researchers should implement multi-level experimental approaches. Western blot analysis should focus on key phosphorylated proteins in this pathway, including p-EGFR (Tyr1068), p-AKT (Ser473), and p-mTOR (Ser2448), comparing control vs. KBTBD7-knockdown cells . Time-course experiments following EGF stimulation can reveal dynamic changes in pathway activation. RT-PCR should be used to distinguish between transcriptional and post-translational effects on pathway components, as research has shown KBTBD7 affects EGFR protein levels but not mRNA expression . Rescue experiments are crucial - the reversal of pathway inhibition through targeted suppression of PTEN in KBTBD7-knockdown cells provides strong evidence for the KBTBD7-PTEN-EGFR/PI3K/AKT mechanism . For more detailed analysis, researchers can employ phospho-specific protein arrays to comprehensively profile kinase activities across multiple signaling nodes simultaneously. Proximity ligation assays or FRET-based biosensors can provide spatial information about pathway component interactions and activations within intact cells.

How should researchers design experiments to validate KBTBD7 as a therapeutic target in NSCLC?

Validation of KBTBD7 as a therapeutic target in NSCLC requires a multi-stage experimental approach. Initial validation should expand on the established correlation between KBTBD7 expression and clinicopathological features (histological type, P-TNM stage, lymph node metastasis, tumor size) using larger, diverse patient cohorts with extended follow-up data to assess prognostic value. In vitro studies should compare multiple NSCLC cell lines with varying KBTBD7 expression levels, employing both genetic approaches (siRNA, shRNA, CRISPR) and pharmacological tools (if available) to inhibit KBTBD7. Beyond proliferation and invasion assays, researchers should assess effects on apoptosis, cell cycle progression, and response to standard chemotherapeutics and EGFR-targeted drugs. In vivo validation is essential, using xenograft models with inducible KBTBD7 knockdown systems to demonstrate tumor growth inhibition. Patient-derived xenograft (PDX) models would provide more clinically relevant validation. Target engagement studies should confirm the molecular mechanism, demonstrating that therapeutic KBTBD7 inhibition restores PTEN levels and suppresses EGFR/PI3K/AKT signaling in both cell models and tumor tissues. Finally, predictive biomarker studies should identify patient subgroups most likely to benefit from KBTBD7-targeted therapies.

What are the most appropriate positive and negative controls for KBTBD7 immunohistochemistry in tissue microarrays?

For robust KBTBD7 immunohistochemistry in tissue microarrays, researchers should implement a comprehensive control strategy. Positive controls should include NSCLC tissues previously confirmed to express high KBTBD7 levels, particularly adenocarcinoma samples which have shown stronger KBTBD7 positivity than squamous cell carcinoma . Including cell line pellets with known KBTBD7 expression (such as A549 or H1299) embedded in the tissue microarray provides additional standardized positive controls. For negative controls, paired adjacent non-cancerous lung tissues from the same patients offer the most relevant comparison, as these have consistently shown lower KBTBD7 expression than tumor tissues . Technical negative controls should include primary antibody omission and isotype controls using irrelevant antibodies of the same isotype and concentration. For advanced validation, tissues from experimental models with KBTBD7 knockdown or knockout can serve as specificity controls. To address staining variability, the microarray should include internal reference tissues that maintain consistent KBTBD7 expression across experiments. Standardized scoring criteria, as described in published research (combined intensity and area scores of 0-12, with >6 considered positive) , should be employed with multiple independent pathologists scoring blindly.

How can researchers effectively combine KBTBD7 antibody-based techniques with RNA analysis for comprehensive protein function studies?

For comprehensive analysis of KBTBD7 function, researchers should strategically integrate protein and RNA-level investigations. Parallel protein detection (via Western blot, immunohistochemistry, or immunofluorescence) and mRNA quantification (via RT-PCR or RNA-seq) can distinguish between transcriptional and post-translational regulation, as demonstrated in KBTBD7 studies showing protein-level effects on EGFR without corresponding mRNA changes . For spatial correlation, researchers can employ RNAscope in situ hybridization alongside immunohistochemistry on consecutive tissue sections to visualize KBTBD7 mRNA and protein distribution patterns. Single-cell approaches combining protein (CyTOF) and RNA (scRNA-seq) analyses can reveal cell type-specific expression patterns. To investigate KBTBD7's post-translational regulatory functions, researchers should examine both KBTBD7 protein levels and the expression/activity of its targets (PTEN, TIAM1, DRD2) along with downstream effectors (EGFR, PI3K/AKT pathway components) . For mechanistic studies, KBTBD7 RNA interference should be complemented with protein-level rescue experiments and ubiquitination assays. Time-course experiments following KBTBD7 manipulation can reveal the temporal relationship between RNA changes, protein alterations, and functional outcomes, providing insight into the regulatory hierarchy.

How should researchers address inconsistent KBTBD7 antibody staining in immunohistochemistry applications?

When facing inconsistent KBTBD7 antibody staining in immunohistochemistry, researchers should systematically troubleshoot pre-analytical, analytical, and post-analytical variables. Pre-analytical factors to examine include tissue fixation time (standardize to 24-48 hours in 10% neutral buffered formalin), tissue processing protocols, and storage conditions of paraffin blocks and slides. For KBTBD7 specifically, antigen retrieval optimization is critical - EDTA-based retrieval at high temperature for 20 minutes has proven effective , but pH and buffer composition may need adjustment for different tissue types. Analytically, antibody concentration should be titrated (1:100 dilution has worked for KBTBD7 ), incubation time standardized, and detection systems validated. If inconsistency persists, try alternative KBTBD7 antibody clones targeting different epitopes, as conformational changes might affect accessibility in certain samples. For interpretation, implement the standardized scoring system used in successful studies (intensity scale 0-3, area scale 1-4, with final scores >6 considered positive) . Multi-observer blinded scoring reduces subjective bias. When comparing across sample batches, always include internal reference controls. If discrepancies occur between antibody lots, perform parallel validation with alternative detection methods like Western blotting using the same samples to confirm expression patterns.

How can KBTBD7 antibodies be effectively used in patient stratification for potential targeted therapies?

For effective patient stratification using KBTBD7 antibodies, researchers should develop a standardized immunohistochemical protocol with clearly defined positivity thresholds based on the established scoring system (combined intensity/area scores >6 indicating positivity) . This approach should be validated in large retrospective cohorts correlating KBTBD7 expression with treatment outcomes and clinicopathological features. Multiplex immunohistochemistry or immunofluorescence techniques should be explored to simultaneously assess KBTBD7 alongside other relevant markers in the EGFR/PI3K/AKT pathway (PTEN, phosphorylated EGFR, AKT, mTOR) , providing a more comprehensive stratification approach. Researchers should investigate whether KBTBD7 expression patterns can predict response to existing therapies targeting the EGFR/PI3K/AKT pathway, potentially identifying patients who might benefit from combination approaches addressing both KBTBD7 and downstream effectors. For clinical implementation, automated image analysis algorithms should be developed to standardize KBTBD7 scoring, reducing inter-observer variability. Ultimately, prospective clinical studies will be needed to validate cutoff values and determine whether KBTBD7-based stratification improves treatment outcomes. Complementary liquid biopsy approaches, if KBTBD7 protein or its surrogate markers can be detected in circulation, might offer less invasive monitoring options.

What experimental design is most appropriate for evaluating KBTBD7 as a biomarker in longitudinal NSCLC studies?