INTS14 Antibody

What is INTS14 and what is its role in cellular processes?

INTS14, also known as von Willebrand factor A domain containing 9 (VWA9) or Chromosome 15 open reading frame 44 (C15orf44), functions as a subunit of the Integrator complex involved in the transcription and 3'-box-dependent processing of small nuclear RNAs (snRNAs) U1 and U2 . INTS14 forms part of a modular extension of the core Integrator complex alongside INTS13 (Asunder) and INTS10 . Recent research has identified INTS14 as a component of a tetrameric "Arm module" with INTS13, INTS10, and INTS15 (previously known as C7ORF26) . In humans, the canonical INTS14 protein consists of 518 amino acid residues, has a molecular mass of 57.5 kDa, and localizes primarily to the nucleus .

What applications are INTS14 antibodies validated for?

INTS14 antibodies are validated for multiple research applications including immunohistochemistry (IHC), immunocytochemistry with immunofluorescence (ICC-IF), and Western blotting (WB) . For Western blotting applications, the recommended concentration range is 0.04-0.4 μg/mL, while immunohistochemistry typically requires a dilution range of 1:50-1:200 . Additionally, enzyme-linked immunosorbent assay (ELISA) represents another common application for INTS14 antibodies in research settings .

What species do commercially available INTS14 antibodies react with?

Commercial INTS14 antibodies demonstrate reactivity with human, mouse, and rat species . For example, the polyclonal antibody against INTS14 (HPA040651) is specifically designed against human INTS14 but exhibits cross-reactivity with mouse and rat orthologs . INTS14 gene orthologs have been identified across multiple species including mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken , suggesting potential cross-reactivity with these species, though specific validation would be required for confirmation.

How does INTS14 interact with other components of the Integrator complex?

INTS14 forms a stable complex with INTS13, INTS10, and INTS15, collectively comprising the "Arm module" of the Integrator complex . The interaction between INTS14 and INTS10 specifically involves the C-terminal region of the INTS10 helical repeats . Mutations in INTS10 residues within this region, particularly E633A and E633A/E634A, result in disrupted INTS14 recruitment, supporting computational interaction models .

The MIDAS (Metal Ion-Dependent Adhesion Site) pocket of the INTS14 VWA domain plays a crucial role in INTS10 recruitment . Specific mutations in INTS14 residues (D8A/S10A/S12A and L11E/R15A) disrupt binding with INTS10 . These mutations align with the interface predicted by structural modeling, confirming this region's importance for protein-protein interaction .

The Arm module connects to the core Integrator complex through an interaction between INTS15 and the INTS5/8 subcomplex (the "Shoulder module") . Size-exclusion chromatography demonstrates that combining INTS13/14/10/15 with INTS5/8 produces a high molecular weight complex containing all six proteins, while the same experiment without INTS15 shows no interaction . This confirms INTS15's critical role in tethering the Arm module to the core Integrator complex .

What mutations in INTS14 affect its binding with INTS10 and how can antibodies help study these interactions?

Mutations in the MIDAS pocket of INTS14's VWA domain, specifically D8A/S10A/S12A and L11E/R15A, disrupt INTS10 binding . These mutations are located precisely at the predicted interface between the two proteins . The table below summarizes key mutations and their effects:

INTS14 antibodies facilitate the study of these interactions through co-immunoprecipitation experiments, where wild-type and mutant proteins are expressed in cells, precipitated using antibodies, and their interactions analyzed through immunoblotting . This approach allows researchers to validate computational models and identify critical residues for protein-protein interactions.

How can researchers use INTS14 antibodies to investigate structural relationships within the Integrator complex?

INTS14 antibodies serve as powerful tools for investigating the structural organization of the Integrator complex through several approaches:

• Affinity purification coupled with mass spectrometry: Using INTS14 antibodies to isolate native complexes followed by LC-MS/MS analysis has helped identify all Integrator subunits, including the previously uncharacterized INTS15 .

• Glycerol gradient fractionation: After affinity purification with INTS14 antibodies, subjecting eluates to glycerol gradient centrifugation helps determine the association of INTS14 with smaller modules versus the complete Integrator complex .

• Salt stability assays: Performing affinity purification under increasing salt concentrations (up to 750 mM KCl) helps assess the stability of INTS14's associations with other complex components .

• Reconstitution experiments: Using recombinant proteins and INTS14 antibodies to validate direct interactions between complex components and their subcomplexes provides insights into the modular architecture of the Integrator complex .

These approaches have revealed that INTS14 primarily associates with the smaller INTS13/14/10/15 module rather than the complete Integrator complex, suggesting a modular organization with functional implications .

Western Blotting Protocol:

• Recommended concentration: 0.04-0.4 μg/mL

• Sample preparation: Standard protein extraction from cells/tissues

• Separation: SDS-PAGE under reducing conditions

• Transfer: To PVDF or nitrocellulose membrane

• Blocking: 5% non-fat milk or BSA in TBST (1 hour, room temperature)

• Primary antibody: Apply INTS14 antibody at recommended concentration in blocking buffer (overnight, 4°C)

• Washing: 3-5 times with TBST

• Secondary antibody: HRP-conjugated anti-rabbit IgG (1:5000-1:10000, 1 hour, room temperature)

• Detection: Enhanced chemiluminescence

Immunohistochemistry Protocol:

• Recommended dilution: 1:50-1:200

• Sample preparation: Formalin-fixed, paraffin-embedded sections

• Deparaffinization and rehydration

• Antigen retrieval: Heat-induced in citrate buffer (pH 6.0)

• Blocking: 5-10% normal serum in PBS (1 hour, room temperature)

• Primary antibody: Apply INTS14 antibody at recommended dilution (overnight, 4°C)

• Washing: 3 times with PBS

• Secondary antibody: Biotinylated or polymer-based detection system

• Visualization: DAB or similar chromogen

• Counterstaining: Hematoxylin

How can researchers validate the specificity of INTS14 antibodies?

Validating INTS14 antibody specificity is critical for ensuring reliable experimental results. Researchers should implement the following validation approaches:

• Genetic knockdown/knockout controls: Compare antibody signals in wild-type cells versus INTS14-depleted cells (using siRNA, shRNA, or CRISPR-Cas9). A specific antibody will show significantly reduced signal in knockdown/knockout samples.

• Overexpression controls: Compare signals between control cells and those overexpressing tagged INTS14. A specific antibody will show enhanced signal intensity in overexpressing cells.

• Peptide competition: Pre-incubate the antibody with the immunizing peptide or recombinant INTS14 protein before application. Specific binding should be blocked, resulting in signal reduction.

• Multi-antibody validation: Use antibodies targeting different INTS14 epitopes and confirm consistent results.

• Mass spectrometry validation: Perform immunoprecipitation with the INTS14 antibody followed by mass spectrometry analysis to confirm INTS14 detection among precipitated proteins .

What experimental controls should be included when working with INTS14 antibodies?

When designing experiments with INTS14 antibodies, researchers should incorporate these essential controls:

Negative Controls:

• Primary antibody omission control

• Isotype control (irrelevant antibody of same isotype and concentration)

• Blocking peptide competition

• Cell lines with confirmed low/no INTS14 expression

Positive Controls:

• Cancer cell lines with high INTS14 expression (lung, prostate, colon, stomach, skin cancer cells)

• Recombinant INTS14 protein (if available)

• Tissues with confirmed INTS14 expression

Technical Controls:

• Loading controls for Western blotting (GAPDH, β-actin, total protein staining)

• Antibody titration to determine optimal concentration

• Processing controls for consistency across samples

Validation Controls:

• INTS14 knockdown/knockout samples

• INTS14 overexpression samples

• Multiple antibodies targeting different epitopes

Is INTS14 expression altered in cancer cells and how can antibodies quantify these changes?

INTS14 exhibits significantly elevated expression in multiple cancer types compared to corresponding non-cancerous tissues, including lung, prostate, colon, stomach, and skin cancers . INTS14 antibodies enable quantitative analysis of this differential expression through:

• Immunohistochemical analysis of tissue microarrays containing paired tumor and normal samples, allowing for scoring of staining intensity and percentage of positive cells.

• Western blot analysis with densitometric quantification to compare INTS14 protein levels across different cell lines and tissue samples.

• Immunofluorescence microscopy to assess both expression levels and subcellular localization changes in cancer cells.

• Flow cytometry with permeabilized cells to quantify INTS14 expression at the single-cell level across different cancer subtypes.

These antibody-based approaches provide complementary data to transcript-level analyses, offering insights into post-transcriptional regulation of INTS14 in cancer progression.

How can INTS14 antibodies be used to investigate its role in RNA processing dysfunctions?

As a component of the Integrator complex involved in snRNA processing, INTS14 may contribute to RNA processing dysfunctions in disease states. Researchers can use INTS14 antibodies to investigate this through:

• RNA immunoprecipitation (RIP): Using INTS14 antibodies to pull down INTS14-associated RNA complexes, followed by RT-qPCR or sequencing to identify bound RNAs and how their interactions change in disease conditions.

• Chromatin immunoprecipitation (ChIP): Employing INTS14 antibodies to identify genomic regions where the Integrator complex is recruited, particularly at snRNA genes and other transcriptionally active sites.

• Proximity ligation assays (PLA): Utilizing INTS14 antibodies in combination with antibodies against other RNA processing factors to visualize and quantify protein-protein interactions within the RNA processing machinery.

• Immunoprecipitation-mass spectrometry: Using INTS14 antibodies to isolate protein complexes from normal and diseased tissues to identify altered protein interactions in disease states.

These approaches can reveal how INTS14 dysfunction might contribute to aberrant RNA processing in cancer and other diseases, potentially identifying new therapeutic targets within this pathway.

How can INTS14 antibodies facilitate research on the newly discovered INTS13/14/10/15 "Arm module"?

The recent identification of INTS15 as a component of the INTS13/14/10 module opens new research avenues where INTS14 antibodies can play crucial roles:

• Structural characterization: INTS14 antibodies can help validate protein-protein interactions predicted by structural modeling, particularly at the interfaces between INTS14 and other module components.

• Functional analysis: By immunodepleting INTS14 from nuclear extracts or using antibodies to disrupt specific interactions, researchers can assess the functional consequences of Arm module perturbation on RNA processing.

• Dynamic assembly studies: Using INTS14 antibodies in combination with different extraction conditions and biochemical fractionation techniques can reveal how the Arm module assembles with the core Integrator complex under different cellular conditions.

INTS14 antibodies thus provide essential tools for understanding both the structural organization and functional significance of this newly characterized module within the larger Integrator complex architecture .

What methodological advances might improve INTS14 antibody applications in research?

Several methodological advances could enhance INTS14 antibody applications:

• Development of monoclonal antibodies: While polyclonal antibodies against INTS14 are currently available , developing monoclonal antibodies targeting specific epitopes could improve consistency and specificity.

• Proximity-dependent labeling: Conjugating INTS14 antibodies with enzymes like BioID or APEX2 would allow identification of proteins in close proximity to INTS14 in living cells.

• Super-resolution microscopy compatible antibodies: Developing directly conjugated INTS14 antibodies optimized for techniques like STORM or PALM would enable nanoscale visualization of INTS14 within nuclear complexes.

• Intrabodies: Engineering antibody fragments that recognize INTS14 and function within living cells could enable real-time tracking and perturbation of INTS14 function.

These advances would expand the toolkit available for investigating INTS14's role in the Integrator complex and RNA processing pathways in both normal and disease states.