KBTBD4 Antibody

What is KBTBD4 and what is its relevance in cancer research?

KBTBD4 is a substrate receptor of the CULLIN3-RING E3 ubiquitin ligase complex that plays a critical role in protein degradation pathways. In humans, the canonical protein has a length of 518 amino acid residues with a molecular mass of 58.1 kDa . KBTBD4 has gained significant attention in cancer research due to its recurrent mutations in medulloblastoma, the most common malignant brain tumor in children . These mutations create neomorphic protein-protein interactions that drive aberrant function, making KBTBD4 a potential therapeutic target for certain medulloblastoma subtypes.

What are the structural domains of KBTBD4 and how do they contribute to antibody epitope selection?

KBTBD4 contains two key structural domains:

• BTB (Broad-Complex, Tramtrack, and Bric-a-brac) domain: Mediates interaction with CULLIN3 in the E3 ligase complex

• Kelch repeat domain: Forms a β-propeller structure involved in substrate recognition

When selecting antibodies, researchers should consider whether their experimental goals require detection of specific domains. For studying KBTBD4's interaction with the CULLIN3 complex, antibodies targeting the BTB domain may be preferable. For investigating substrate interactions, antibodies recognizing the Kelch repeat domain would be more appropriate. Disease-associated mutations frequently occur in the Kelch domain region, so researchers studying medulloblastoma may need antibodies that can distinguish between wild-type and mutant forms.

How do KBTBD4 isoforms affect antibody selection for research applications?

Up to two different isoforms have been reported for KBTBD4 , which presents important considerations for antibody selection:

• For experiments requiring detection of all KBTBD4 isoforms, select antibodies targeting conserved regions present across all variants.

• For isoform-specific detection, choose antibodies raised against unique sequence regions.

• When interpreting experimental results, be aware that different antibodies may detect distinct subsets of KBTBD4 isoforms, potentially leading to apparently contradictory results.

Methodologically, validate antibody specificity using overexpression systems with tagged isoform-specific constructs, or use siRNA targeting specific isoforms to confirm antibody selectivity.

What mutations in KBTBD4 are associated with medulloblastoma, and how might this affect antibody-based detection?

Medulloblastoma-associated KBTBD4 mutations include recurrent in-frame insertions that create gain-of-function alterations. Two specific mutations have been well-characterized:

• KBTBD4-P311PP: Insertion of a proline at position 311

• KBTBD4-R313PRR: Insertion of proline and arginine at position 313

These mutations present unique challenges for antibody-based detection:

When studying these mutations, researchers should validate antibody performance with both wild-type and mutant KBTBD4 expression constructs to ensure reliable detection of both forms.

How do KBTBD4 mutations affect its interaction with substrates, and how can antibodies help study this?

KBTBD4 mutations promote neomorphic substrate interactions through the following mechanism:

• Mutant KBTBD4 (P311PP and R313PRR) gains the ability to interact with HDAC1/2 in the CoREST complex .

• This interaction leads to ubiquitination and degradation of CoREST and LSD1, while wild-type KBTBD4 lacks this activity .

• The degradation of CoREST relieves transcriptional repression of its target genes, promoting a stem-like signature in cancer cells .

To study these interactions using antibodies:

• Perform co-immunoprecipitation with anti-KBTBD4 antibodies followed by detection of CoREST, LSD1, and HDAC1/2.

• Use reciprocal co-immunoprecipitation with antibodies against CoREST complex components.

• Include controls with neddylation inhibitors (MLN4924) to prevent ubiquitylation activity of CRL3 when studying transient interactions .

What experimental approaches using antibodies can help validate KBTBD4 as a therapeutic target?

To validate KBTBD4 as a therapeutic target using antibody-based approaches:

• Target validation in patient-derived models:

  • Use immunoblotting with anti-KBTBD4 antibodies to confirm expression in patient-derived xenograft (PDX) models.

  • Assess CoREST and LSD1 levels by immunoblotting after KBTBD4 inactivation through base editing, as demonstrated in ICB1299 PDX cells .

• Therapeutic intervention assessment:

  • Monitor CoREST and LSD1 protein levels by immunoblotting after treatment with potential inhibitors.

  • For example, ex vivo treatment of KBTBD4-PR mutant PDX cells with MLN4924 and HDAC1/2 inhibitor RBC1HI led to increased levels of CoREST and LSD1, consistent with KBTBD4 inhibition .

• Mechanism of action studies:

  • Use time-resolved Förster resonance energy transfer (TR-FRET) assays to measure the association between KBTBD4 and the LSD1-HDAC1-CoREST (LHC) complex .

  • Assess how potential therapeutic compounds affect this interaction.

What criteria should researchers use when selecting anti-KBTBD4 antibodies for specific applications?

When selecting anti-KBTBD4 antibodies, consider these application-specific criteria:

Additionally, consider whether your research requires:

• Detection of specific mutations (P311PP or R313PRR)

• Ability to distinguish between phosphorylated and non-phosphorylated forms

• Cross-reactivity with orthologs if working with model organisms

How can researchers validate the specificity of anti-KBTBD4 antibodies?

A comprehensive validation strategy for anti-KBTBD4 antibodies includes:

• Genetic validation:

  • Use CRISPR/Cas9 knockout or siRNA knockdown of KBTBD4

  • Apply base editing approaches as demonstrated in PDX models to introduce hypomorphic mutations (e.g., Y120H, C297R, or M316T/W317R in KBTBD4-PR)

• Expression validation:

  • Compare antibody detection in cell lines with varying endogenous KBTBD4 levels

  • Test medulloblastoma cell lines of different subgroups that express KBTBD4 (e.g., D283Med, HD-MB03, D425Med, D458Med, and DAOY)

• Specificity controls:

  • Perform peptide competition assays with the immunizing antigen

  • Compare detection patterns between multiple antibodies targeting different KBTBD4 epitopes

What methodological approaches enable optimal protein extraction for KBTBD4 detection?

KBTBD4 is a component of ubiquitin ligase complexes, requiring specialized extraction methods:

• Lysis buffer optimization:

  • Use buffers containing 1% NP-40 or Triton X-100 with 150-300 mM NaCl

  • Include deubiquitinase inhibitors (e.g., PR-619, 1,10-phenanthroline)

  • Add proteasome inhibitors (MG132) to prevent degradation of ubiquitinated proteins

• Preserving protein interactions:

  • For studying KBTBD4-CoREST interactions, include InsP6 (inositol hexakisphosphate), which stabilizes the interaction between HDAC1/2 and CoREST

  • Consider mild crosslinking (0.1-0.5% formaldehyde) before lysis to stabilize transient interactions

• Subcellular fractionation:

  • Separate nuclear and cytoplasmic fractions to assess KBTBD4 distribution

  • Include phosphatase inhibitors to preserve phosphorylation status

How can researchers effectively use anti-KBTBD4 antibodies to study protein-protein interactions?

To investigate KBTBD4 protein interactions:

• Co-immunoprecipitation (Co-IP):

  • Immunoprecipitate KBTBD4 using specific antibodies and detect interacting partners

  • Include appropriate controls: IgG pull-downs and conditions that disrupt expected interactions

  • Example: Co-IP experiments with HA–KBTBD4-PR efficiently retrieved LSD1, CoREST, HDAC1, and HDAC2

• Proximity-based approaches:

  • Combine with proximity ligation assay (PLA) to visualize KBTBD4-substrate interactions in situ

  • Use GST-fused ubiquitin binding domains (UBA) to isolate ubiquitylated proteins interacting with KBTBD4

• Functional validation:

  • Compare interactions between wild-type and mutant KBTBD4

  • Use neddylation inhibitors (MLN4924) to prevent the ubiquitylation activity of CRL3 when studying transient interactions

What protocols enable researchers to study KBTBD4-mediated ubiquitination using antibodies?

To investigate KBTBD4-mediated ubiquitination:

• Cellular ubiquitination assays:

  • Immunoprecipitate KBTBD4 or its substrates (e.g., CoREST)

  • Detect ubiquitination using anti-ubiquitin antibodies

  • Example: "KBTBD4 R313PRR and KBTBD4 P311PP promoted ubiquitylation of endogenous CoREST, while ubiquitylation of CoREST in parental D283Med cells or in the presence of KBTBD4 WT was undetectable"

• In vitro ubiquitination reconstitution:

  • Use purified components to reconstitute CRL3-KBTBD4 ubiquitination system

  • Compare activities of wild-type and mutant KBTBD4

  • Example: "Reconstituted CRL3 KBTBD4-PR exhibited increased ubiquitination of CoREST in vitro in comparison with CRL3 KBTBD4-WT"

• Substrate stabilization:

  • Use proteasome inhibitors (MG132) to prevent degradation of ubiquitinated proteins

  • Compare substrate levels with and without inhibitors to assess degradation efficiency

How can researchers use anti-KBTBD4 antibodies to analyze its expression in tumor samples?

For analyzing KBTBD4 expression in tumor samples:

• Immunohistochemistry optimization:

  • Test multiple fixation and antigen retrieval methods

  • Validate antibody specificity using positive controls (cell lines with known KBTBD4 expression) and negative controls (KBTBD4 knockdown tissues)

  • Consider multiplexed staining with antibodies against CoREST and LSD1 to correlate KBTBD4 with substrate levels

• Patient sample analysis:

  • Compare KBTBD4 levels across medulloblastoma subgroups (WNT, SHH, Group 3, Group 4)

  • Assess correlation between KBTBD4 mutations and protein expression levels

  • Correlate KBTBD4 expression with patient outcomes using tissue microarrays

• PDX model characterization:

  • Use immunoblotting to characterize KBTBD4, CoREST, and LSD1 levels in PDX models

  • Example: Global proteomics comparing wild-type and KBTBD4-PR mutant PDX models identified significant changes in the expression of HDAC2-associated proteins, including CoREST complex members

How can researchers distinguish between wild-type and mutant KBTBD4 using antibody-based methods?

Distinguishing between wild-type and mutant KBTBD4 requires specialized approaches:

• Mutation-specific antibodies:

  • Develop antibodies specifically recognizing the insertion mutations (P311PP or R313PRR)

  • Validate using cells expressing either wild-type or mutant KBTBD4

• Functional readouts:

  • Use the differential effect on substrate degradation as a proxy

  • Monitor CoREST and LSD1 levels, which are specifically degraded by mutant KBTBD4

  • Example: "At comparable expression levels of KBTBD4 WT, KBTBD4 R313PRR and KBTBD4 P311PP we observed a reduction in the levels of CoREST only upon expression of KBTBD4 mutants"

• Biochemical separation:

  • Use high-resolution SDS-PAGE to detect subtle size differences

  • Employ 2D gel electrophoresis to separate based on both size and charge

  • Consider mass spectrometry following immunoprecipitation to identify mutation-specific peptides

What antibody-based approaches can identify novel substrates of mutant KBTBD4?

To identify novel substrates of mutant KBTBD4:

• Quantitative proteomics:

  • Compare proteome changes in cells expressing wild-type versus mutant KBTBD4

  • Example: "Comparison of the WT and PR mutant models identified 64 and 82 proteins that were significantly up- and downregulated, respectively, in the mutant samples"

  • Focus on proteins showing reduced levels in mutant KBTBD4-expressing cells

• Immunoprecipitation-mass spectrometry:

  • Use anti-KBTBD4 antibodies to pull down KBTBD4 complexes

  • Identify co-precipitating proteins by mass spectrometry

  • Compare binding partners between wild-type and mutant KBTBD4

  • Include proteasome inhibitors to stabilize substrate interactions

• Ubiquitinome analysis:

  • Enrich for ubiquitinated proteins using anti-ubiquitin antibodies or ubiquitin-binding domains

  • Compare ubiquitination patterns between wild-type and mutant KBTBD4-expressing cells

  • Validate candidates by assessing their degradation kinetics

How can researchers use antibodies to evaluate potential therapeutics targeting mutant KBTBD4?

To evaluate KBTBD4-targeting therapeutics:

• Target engagement assays:

  • Use cellular thermal shift assay (CETSA) with KBTBD4 antibodies to assess compound binding

  • Monitor changes in KBTBD4-substrate interactions using co-immunoprecipitation

  • Example: HDAC1/2 inhibitors can block the mutant KBTBD4-HDAC1 interface

• Functional readouts:

  • Assess CoREST and LSD1 degradation as pharmacodynamic markers

  • Example: "Ex vivo treatment of ICB1572 PDX cells with MLN4924 and RBC1HI, separately, led to increased levels of CoREST and LSD1, consistent with KBTBD4 inhibition"

  • Monitor stem-cell signature gene expression changes by RT-qPCR

• Combination therapy assessment:

  • Use antibody-based readouts to evaluate synergy between KBTBD4-targeting compounds and standard treatments

  • Example: KBTBD4-PR mutant PDX cells showed heightened sensitivity to HDAC1/2 inhibitor RBC1HI compared to KBTBD4-WT cells

How should researchers address inconsistent KBTBD4 detection in different experimental systems?

When facing inconsistent KBTBD4 detection:

• Antibody validation:

  • Verify antibody specificity using genetic controls (knockout/knockdown)

  • Test multiple antibodies targeting different epitopes

  • Consider that different fixation methods may affect epitope availability

• Technical optimization:

  • For weak signals: Test enrichment methods (immunoprecipitation before Western blotting)

  • For multiple bands: Determine if these represent isoforms, post-translational modifications, or degradation products

  • For inconsistent results between applications: Some antibodies work well for Western blotting but poorly for immunoprecipitation

• Biological interpretation:

  • Consider that KBTBD4 expression varies across cell types and medulloblastoma subgroups

  • Example: KBTBD4 has been studied in various medulloblastoma cell lines including D283Med (group 3/4), HD-MB03 (group 3), D425Med (group 3), D458Med, and DAOY (SHH subgroup)

What controls are essential when using anti-KBTBD4 antibodies to study substrate degradation?

Essential controls for KBTBD4-mediated degradation studies:

• Expression controls:

  • Compare effects at equivalent expression levels of wild-type and mutant KBTBD4

  • Example: "At comparable expression levels of KBTBD4 WT, KBTBD4 R313PRR and KBTBD4 P311PP we observed a reduction in the levels of CoREST only upon expression of KBTBD4 mutants"

  • Test dose-dependent effects: "Minimal induction of KBTBD4 P311PP with the lowest doxycycline dose still promoted CoREST degradation. By contrast, the highest expression of KBTBD4 WT did not alter CoREST levels"

• Pathway controls:

  • Include proteasome inhibitors (MG132) to confirm degradation mechanism

  • Use neddylation inhibitors (MLN4924) to prevent CRL3 activation

  • Example: "We detected interaction of CoREST and LSD1 with KBTBD4 R313PRR and KBTBD4 P311PP exclusively in cells treated with MLN4924 where the ubiquitylation activity of CRL3 was prevented"

• Specificity controls:

  • Monitor unrelated proteins to confirm specificity of degradation

  • Include substrate mutants resistant to ubiquitination to validate mechanism

How can researchers interpret contradictory findings about KBTBD4 function between different experimental models?

When resolving contradictory findings about KBTBD4:

• Model-specific considerations:

  • Different medulloblastoma subtypes may show distinct KBTBD4 functions

  • Example: "In terms of transcriptional identity, D283Med are close to the identified subtype II of a more recent medulloblastoma classification of group 3 and 4 subtypes"

  • Cell line genetic background may influence KBTBD4 activity

• Context-dependent activity:

  • KBTBD4 function may depend on the expression of cofactors or substrates

  • Example: KBTBD4-mutant and LHC binding required the addition of inositol hexakisphosphate (InsP6), a cofactor that stabilizes the PPI between HDAC1/2 and CoREST

• Technical reconciliation:

  • Standardize experimental conditions across models

  • Validate findings in clinically relevant models: "Transcriptional analysis of >200 human group 3 and 4 medulloblastomas by RNA-seq, highlights the presence of CoREST and stem-like signatures in tumours with KBTBD4 mutations"