INTS6 Antibody, FITC conjugated

What is INTS6 and what are its primary cellular functions?

INTS6, also known as Int6, DBI-1, DDX26, or DICE1, functions as a component of the Integrator (INT) complex that is critically involved in the transcription and 3'-box-dependent processing of small nuclear RNAs (snRNAs) U1 and U2. The Integrator complex associates with the C-terminal domain (CTD) of RNA polymerase II largest subunit (POLR2A) and is recruited to the U1 and U2 snRNA genes . Additionally, INTS6 mediates recruitment of cytoplasmic dynein to the nuclear envelope, likely as a component of the INT complex . Importantly, INTS6 may function as a tumor suppressor, as ectopic expression has been shown to suppress tumor cell growth across multiple studies .

What are the recommended applications for FITC-conjugated INTS6 antibodies?

FITC-conjugated INTS6 antibodies are primarily recommended for ELISA applications with a suggested dilution range of 1:100-1:500 . The fluorescent conjugation makes this antibody particularly valuable for applications requiring direct visualization without secondary antibodies. While the commercial FITC-conjugated antibody is specifically validated for ELISA, researchers should consider that fluorescently labeled antibodies may also be suitable for flow cytometry and immunofluorescence microscopy after proper validation, particularly when analyzing the expression and localization of INTS6 in cell populations or tissue sections.

What validation methods should be employed before using INTS6 antibodies in experiments?

When validating INTS6 antibodies for research use, a multi-tiered approach is recommended:

• Specificity testing: Compare antibody binding in wild-type cells versus INTS6 knockout or knockdown cells to confirm specific recognition

• Western blot validation: Verify that the antibody detects a band of appropriate molecular weight (approximately 100 kDa for INTS6)

• Cross-reactivity assessment: Test the antibody against related proteins or in multiple cell types

• Positive and negative controls: Include tissues or cells known to express high or low levels of INTS6

• Concentration optimization: Perform titration experiments to determine optimal working dilution (1:100-1:500 for ELISA applications with the FITC-conjugated antibody)

How should sample preparation be optimized for INTS6 detection using FITC-conjugated antibodies?

Optimal sample preparation for INTS6 detection requires consideration of several factors:

When using FITC-conjugated antibodies, it's crucial to minimize exposure to light throughout the protocol to prevent photobleaching. For optimal results with the FITC-conjugated polyclonal INTS6 antibody, researchers should work with recombinant human INTS6 protein as a positive control, particularly the region spanning amino acids 571-795, which corresponds to the immunogen used to generate the antibody .

How does the INTS6-PP2A interaction regulate transcriptional processes?

Recent research has revealed that INTS6 serves as a critical bridge between the Integrator complex and protein phosphatase 2A (PP2A) . This interaction creates a functional opposition to CDK9-mediated RNA polymerase II (RNAPII) pause-release. Immunoprecipitation studies followed by mass spectrometry have demonstrated that INTS6 co-purifies with PP2A structural subunits (PPP2R1A/PPP2R1B) and the PPP2CA catalytic subunit (PP2A-C), as well as members of the RNAPII complex including Rpb1/POLR2A .

The PP2A-Integrator complex functions to fine-tune transcription by opposing the phosphorylation of RNAPII by CDK9. When INTS6 is depleted, cells demonstrate resistance to CDK9 inhibitors, with RNAPII and phospho-Ser2 RNAPII accumulating genome-wide, particularly across gene bodies of responsive genes . This mechanism reveals INTS6 as a central component in a phosphorylation-dephosphorylation regulatory circuit controlling transcriptional pause-release.

What are the optimal storage and handling conditions for maintaining FITC-conjugated INTS6 antibody activity?

To maintain optimal activity of FITC-conjugated INTS6 antibodies:

• Storage temperature: Store at -20°C for long-term storage and at 4°C for short-term use

• Light protection: FITC is photosensitive; store in amber vials or wrapped in aluminum foil

• Aliquoting: Divide into single-use aliquots to avoid repeated freeze-thaw cycles

• Buffer conditions: Store in phosphate-buffered solution with protein stabilizers and preservatives

• Working dilutions: Prepare fresh working dilutions on the day of use

• Handling: Minimize exposure to strong light sources during experimental procedures

How can INTS6 antibodies be utilized in ChIP-seq experiments to study transcriptional regulation?

ChIP-seq experiments with INTS6 antibodies can provide valuable insights into the genome-wide binding patterns of the Integrator complex and its association with transcriptional machinery. Based on recent research involving INTS6 and transcriptional regulation , the following protocol modifications are recommended:

• Crosslinking optimization: Use 1% formaldehyde for 10 minutes at room temperature, as INTS6 forms protein-protein complexes with both RNAPII and PP2A

• Sonication parameters: Adjust to generate fragments of 200-500 bp for optimal resolution of binding sites

• Antibody selection: For ChIP-seq applications, use non-conjugated antibodies with validated ChIP performance

• Controls: Include input DNA, IgG controls, and where possible, INTS6-depleted cells as negative controls

• Data analysis: Focus on correlation with RNAPII binding sites, particularly at genes regulated by pause-release mechanisms

When analyzing ChIP-seq data, researchers should examine INTS6 enrichment at promoter regions and across gene bodies of actively transcribed genes, as the INTS6-containing PP2A-Integrator complex has been shown to regulate RNAPII phosphorylation and transcriptional pause-release .

What are the mechanistic implications of INTS6 loss in cancer development?

INTS6 has been identified as a potential tumor suppressor, with ectopic expression suppressing tumor cell growth . The mechanistic basis for this tumor suppressor function appears to be multifaceted:

• Transcriptional regulation: As part of the PP2A-Integrator complex, INTS6 helps regulate proper RNAPII pause-release , which may prevent dysregulated expression of oncogenes

• snRNA processing: Disruption of proper snRNA biogenesis through INTS6 loss may lead to aberrant splicing that affects tumor suppressor or oncogene expression

• Cell cycle control: The interaction between INTS6 and PP2A suggests a role in dephosphorylation events that may influence cell cycle progression

• Resistance to CDK9 inhibition: Loss of INTS6 confers resistance to CDK9 inhibitors in cancer cell lines , suggesting altered transcriptional dependencies

Understanding these mechanisms is critical for developing therapeutic strategies targeting transcriptional dysregulation in cancers with altered INTS6 expression or function.

How can FITC-conjugated INTS6 antibodies be used in multiparameter flow cytometry?

Multiparameter flow cytometry using FITC-conjugated INTS6 antibodies requires careful panel design to avoid spectral overlap and ensure accurate detection:

• Panel design considerations:

  • FITC emits in the green spectrum (peak emission ~520 nm)

  • Avoid fluorophores with significant spectral overlap (PE, GFP)

  • Pair with far-red dyes (APC, Alexa Fluor 647) for minimal compensation requirements

• Optimization protocol:

  • Perform single-color controls with each antibody in your panel

  • Use fluorescence-minus-one (FMO) controls to set appropriate gates

  • Titrate the INTS6-FITC antibody (starting with 1:100 dilution) to determine optimal signal-to-noise ratio

• Sample preparation for intracellular staining:

  • Fix cells with 2-4% paraformaldehyde

  • Permeabilize with 0.1% saponin or 0.3% Triton X-100

  • Block with 1-5% BSA before antibody incubation

  • Incubate with INTS6-FITC antibody at optimized concentration

This approach allows researchers to correlate INTS6 expression with other cellular markers, providing insights into how INTS6 expression varies across cell types or cell cycle stages.

What are the most common challenges when working with INTS6 antibodies and how can they be addressed?

How do INTS6 detection methods differ between various experimental techniques?

Different experimental approaches require specific considerations for optimal INTS6 detection:

• ELISA applications: The FITC-conjugated polyclonal antibody is specifically validated for ELISA with recommended dilutions of 1:100-1:500 . This application is ideal for quantifying INTS6 in solution.

• Western blotting: Non-conjugated antibodies like the rabbit polyclonal antibody (ab86369) are recommended for western blot applications . For optimal results, use 20-40 μg of total protein lysate and ensure complete transfer of high molecular weight proteins.

• Immunohistochemistry: Formalin-fixed paraffin-embedded (FFPE) sections have been successfully analyzed using antibodies like ab86369 at 1/200 dilution (1μg/ml) . Antigen retrieval is typically required for optimal staining.

• Immunofluorescence: When using FITC-conjugated antibodies for direct immunofluorescence, minimize photobleaching by reducing exposure to light and using anti-fade mounting media containing DAPI for nuclear counterstaining.

• Co-immunoprecipitation: For studying INTS6 protein interactions, such as with PP2A or RNAPII components, use antibodies validated for immunoprecipitation and gentle lysis conditions to preserve protein complexes .

How can researchers distinguish between INTS6 and other Integrator complex subunits in functional studies?

Distinguishing the specific functions of INTS6 from other Integrator subunits requires targeted approaches:

• Selective knockdown/knockout: Use siRNA or CRISPR-Cas9 to specifically target INTS6 while monitoring other Integrator subunits to ensure specificity

• Rescue experiments: After INTS6 depletion, reintroduce wild-type or mutant INTS6 to identify domains critical for specific functions

• Comparative phenotyping: Compare phenotypes between cells lacking INTS6 versus other Integrator subunits (e.g., INTS8 or INTS11)

• Domain-specific antibodies: Use antibodies targeting unique domains of INTS6 not present in other Integrator subunits

• Protein-protein interaction mapping: Perform immunoprecipitation followed by mass spectrometry to identify INTS6-specific interacting partners not shared with other Integrator subunits

Research has shown that while INTS6 deletion confers resistance to CDK9 inhibition, analogous assays with sgRNAs targeting INTS3 and INTS11 did not confer similar resistance, highlighting the functional specificity of INTS6 within the Integrator complex .

How does INTS6 contribute to the PP2A-Integrator complex's role in transcriptional regulation?

Recent studies have identified INTS6 as a critical bridge between the Integrator complex and the protein phosphatase 2A (PP2A) complex . This interaction creates a functional opposition to CDK9-mediated RNA polymerase II (RNAPII) pause-release. The specific contributions of INTS6 include:

• Physical scaffolding: INTS6 physically connects PP2A components (PPP2R1A/PPP2R1B structural subunits and PPP2CA catalytic subunit) with the Integrator complex

• Targeting to transcriptional machinery: The INTS6-PP2A complex associates with RNAPII, including the Rpb1/POLR2A catalytic core

• Opposing CDK9 activity: INTS6 deletion confers resistance to CDK9 inhibitors, suggesting that INTS6-associated PP2A actively opposes CDK9-mediated phosphorylation

• Fine-tuning gene expression: The INTS6-PP2A-Integrator complex regulates the balance of phosphorylation and dephosphorylation events that control transcriptional pause-release

Understanding this regulatory circuit provides new insights into the molecular mechanisms controlling gene expression and offers potential targets for therapeutic intervention in diseases characterized by dysregulated transcription.

What are the implications of INTS6's tumor suppressor function for cancer research?

The identification of INTS6 as a potential tumor suppressor opens several avenues for cancer research:

• Biomarker development: INTS6 expression levels or mutation status could serve as prognostic or predictive biomarkers in certain cancer types

• Therapeutic targeting: Understanding how INTS6 suppresses tumor growth may reveal vulnerable nodes in cancer cell signaling networks

• Resistance mechanisms: INTS6 loss confers resistance to CDK9 inhibitors , suggesting that INTS6 status might predict response to certain targeted therapies

• Transcriptional dependencies: Cancer cells with altered INTS6 function may exhibit specific transcriptional dependencies that could be therapeutically exploited

• Combination strategies: The connection between INTS6 and the PP2A-Integrator complex suggests potential synergies between phosphatase activators and transcriptional inhibitors

Research into these areas could lead to novel diagnostic tools and therapeutic approaches for cancers characterized by INTS6 dysregulation.