INTS9 Antibody

What is INTS9 and what cellular functions does it perform?

INTS9 is a ~75 kDa protein that serves as a subunit of the Integrator complex, which binds to the C-terminal domain (CTD) of RNA polymerase II . Its primary characterized function is in small nuclear RNA (snRNA) processing, particularly U1 and U2 snRNAs . INTS9 forms a critical heterodimer with INTS11, creating an enzymatically active complex involved in RNA 3'-end processing . Recent research has identified additional roles, including the regulation of neurodevelopmental genes when complexed with BRAT1 . The heterodimer functions in protein-coding gene transcription regulation, with evidence suggesting both enhancement and attenuation of transcription in different contexts .

What antibody options exist for INTS9 detection and which applications are they validated for?

Several commercial antibodies are available for INTS9 detection with distinct application validations:

For optimal ChIP results with CST antibody #13945, use 10 μl of antibody with 10 μg of chromatin (approximately 4 × 10^6 cells) per immunoprecipitation . This antibody has been validated using SimpleChIP Enzymatic Chromatin IP Kits .

How can I validate INTS9 antibody specificity for my research?

Validation should include multiple approaches:

• Western blot with positive control lysates (e.g., Jurkat or HeLa cells) showing the expected 75 kDa band .

• Knockdown/knockout controls using siRNA against INTS9 to demonstrate signal reduction.

• Immunoprecipitation followed by mass spectrometry to confirm pulled-down proteins.

• Testing cross-reactivity against recombinant INTS9 versus related proteins.

• For ChIP applications, include a ChIP-qPCR at known targets of Integrator complex .

The Wu et al. study demonstrated excellent validation by confirming that mutations disrupting INTS9-INTS11 interaction also abolished antibody co-immunoprecipitation, indicating specificity for the properly folded protein .

What are the optimal conditions for Western blotting with INTS9 antibodies?

For optimal Western blot results:

• Sample preparation: Use denaturing lysis buffer as described in Wu et al.

• Protein amount: Load 25-50 μg of total protein per lane

• Antibody dilution: Use 1:1000 dilution for Cell Signaling antibody #13945 or 1:500-1:1000 for ABClonal A10480

• Blocking solution: 5% w/v nonfat dry milk in 1X TBS with 0.1% Tween 20

• Incubation: Overnight at 4°C with gentle shaking

• Detection: Use appropriate HRP-conjugated secondary antibodies and ECL detection systems

Cell Signaling recommends specifically: "For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween 20 at 4°C with gentle shaking, overnight" .

How should I optimize ChIP experiments using INTS9 antibodies?

For successful ChIP experiments with INTS9 antibodies:

• Starting material: Use 4 × 10^6 cells per immunoprecipitation

• Chromatin amount: 10 μg of properly sheared chromatin

• Antibody amount: 10 μl of Cell Signaling #13945 antibody per IP

• Controls: Include IgG negative control and a positive control targeting a known Integrator-bound gene

• Validation: Use ChIP-qPCR to confirm enrichment at known Integrator targets

Based on research by Kirstein et al., INTS9 can be detected at the 5' ends of many protein-coding genes whose expression is regulated by the Integrator complex . When analyzing ChIP-qPCR data for INTS9, expect significant enrichment at promoter regions of genes regulated by Integrator compared to control regions .

What protocol modifications are needed for co-immunoprecipitation of INTS9 with binding partners?

For robust co-immunoprecipitation of INTS9 with its interacting partners:

• Lysis conditions: Use denaturing lysis buffer as described in Wu et al.

• Antibody amount: Use 1:100 dilution of antibody for IP (CST #13945)

• Affinity matrix: Anti-HA affinity resin (for HA-tagged INTS11) or appropriate matrix for your tag system

• Wash conditions: The INTS9-INTS11 heterodimer can withstand rigorous washing with detergent and high salt

• Elution: SDS loading buffer

• Detection: Western blot with appropriate antibodies for suspected binding partners

For studying INTS9-INTS11 interactions specifically, Wu et al. used "myc-tagged IntS9 cDNAs with HA-tagged wild-type IntS11 into 293T cells and subjected the lysates to anti-HA immunoaffinity matrix followed by probing with anti-myc antibodies using Western blot analysis" .

How is the INTS9-INTS11 heterodimer structure organized and what residues are critical for the interaction?

The INTS9-INTS11 C-terminal domain (CTD) complex has been crystallized at 2.1-Å resolution, revealing:

• Structure: A continuous nine-stranded β-sheet (four strands from INTS9 and five from INTS11) with four helices covering one face of the β-sheet

• Interface: Extensive interface between the two CTDs formed by two neighboring strands and two helices

• Critical regions:

  • INTS11 residues 500-600 are essential for interaction with INTS9 CTD

  • Deletion of just 10 residues from the C-terminus of INTS11 (residues 500-590) abolishes the interaction

  • Helix α3 of INTS11 is crucial for the interaction

  • Strand β1 of INTS11 is important for binding

Mutations designed based on this structural information confirmed these critical interaction sites through both yeast two-hybrid assays with the CTDs and co-immunoprecipitation experiments with full-length proteins .

How can I analyze the functional consequences of disrupting the INTS9-INTS11 interaction?

To assess functional impacts of disrupting the INTS9-INTS11 interaction:

• Create point mutations in the interaction interface based on the crystal structure

• Validate disruption of interaction using co-immunoprecipitation in cell culture

• Perform U7 snRNA 3'-end processing assays, as this function is abolished when the interaction is disrupted

• Assess misprocessing of endogenous snRNAs (U2 and U4) using RT-qPCR

• Use RNAi-rescue experiments to confirm the specificity of observed effects

Wu et al. demonstrated that "mutations that disrupt the IntS9–IntS11 interaction also abolish U7 snRNA 3′-end processing, indicating that this interaction is crucial for the function of the Integrator complex" . Their experimental approach included transfecting cells with a U7-GFP reporter and measuring misprocessing through Western blot analysis with antibodies against GFP .

What reagents and expression systems are recommended for recombinant production of the INTS9-INTS11 complex?

For recombinant expression and purification of the INTS9-INTS11 complex:

• Expression vectors:

  • INTS11 CTD (residues 491-600) in pET28a vector with N-terminal His-tag

  • INTS9 CTD (residues 582-658) in pCDFDuet vector without affinity tag

• Co-expression system:

  • E. coli BL21Star (DE3) cells

  • Expression at 23°C for 16-20h

• Purification protocol:

  • Lyse cells by sonication in buffer containing 20 mM Tris (pH 8.5), 200 mM NaCl, and 5% glycerol

  • Purify using Ni-NTA chromatography

  • Remove His-tag with thrombin overnight at 4°C

  • Further purify by gel filtration chromatography (Sephacryl S-300)

  • Concentrate to 30 mg/mL in 20 mM Tris (pH 8.5), 200 mM NaCl, and 10 mM DTT

For crystallization, Wu et al. used "sitting-drop vapor-diffusion method" with "reservoir solution containing 0.1 M Bis-Tris (pH 6.5) and 21–24% (wt/vol) PEG 3350" .

How can I investigate INTS9's role in neuronal development using antibodies?

To study INTS9's role in neuronal development:

• Cell model: Use NT2 cells that can be differentiated into neurons using all-trans retinoic acid (ATRA)

• Experimental approach:

  • Perform ChIP-qPCR to monitor INTS9 occupancy at promoters of neuronal genes before and after differentiation

  • Compare with INTS11 and BRAT1 occupancy patterns

  • Deplete INTS9 using siRNA and assess impact on neuronal differentiation markers

• Gene targets: Focus on neurodevelopmental genes regulated by the INTS9/INTS11/BRAT1 complex

Research by Kirstein et al. showed that "BRAT1 and INTS11 residence at genes induced by ATRA following the differentiation protocol" increased significantly . They also demonstrated that "depletion of BRAT1 led to a significant reduction of INTS11 occupancy" , suggesting interdependent recruitment.

What are the methodological considerations when investigating INTS9 in different protein complexes?

INTS9 participates in multiple distinct complexes, requiring careful experimental design:

• Differential complex identification:

  • Use sequential immunoprecipitation to separate INTS9-INTS11-BRAT1 complex from INTS9-INTS11-INTS4 complex

  • Perform glycerol gradient fractionation to separate complexes by size

• Complex-specific functions:

  • The BRAT1-INTS9-INTS11 complex regulates neurodevelopmental genes

  • The INTS4-INTS9-INTS11 forms the minimal Integrator cleavage module for snRNA processing

  • These complexes are mutually exclusive as BRAT1 and INTS4 compete for binding to INTS9-INTS11

• Disease relevance:

  • Disease-causing mutations of BRAT1 diminish interaction with INTS11/INTS9

  • The E522K mutation in BRAT1 "completely abrogates BRAT1 interaction with INTS11/INTS9"

How can I use INTS9 antibodies to study transcriptional regulation beyond snRNA processing?

For investigating INTS9's wider role in transcriptional regulation:

• Genome-wide approaches:

  • ChIP-seq to map INTS9 binding across the genome

  • Compare with RNA Polymerase II occupancy patterns

  • Integrate with transcriptomic data after INTS9 depletion

• Transcriptional attenuation analysis:

  • Use Precision Run-On sequencing (PRO-seq) to quantify nascent RNA synthesis

  • Focus on promoter-proximal regions where Integrator catalyzes premature termination

  • Examine immediate-early genes where Integrator regulates induction dynamics

• Co-factor interactions:

  • Investigate INTS9 associations with transcriptional elongation factors

  • INTS9/Integrator interacts with NELF, DSIF, and super elongation complex/pTEFb

  • These interactions affect RNAPII pausing and release into productive elongation

Research shows that INTS9 depletion can lead to upregulation of certain genes by preventing premature transcription termination, while in other contexts it may directly promote full-length mRNA production through interaction with elongation factors .

What are common issues when using INTS9 antibodies and how can they be resolved?

Common challenges and solutions when working with INTS9 antibodies:

• Non-specific bands in Western blot:

  • Optimize antibody dilution (start with manufacturer recommendations)

  • Increase blocking time/concentration

  • Use freshly prepared lysates

  • Include appropriate negative controls (INTS9 knockdown)

• Poor ChIP efficiency:

  • Optimize chromatin shearing conditions

  • Increase antibody amount (up to 10 μl per 10 μg chromatin)

  • Extend antibody incubation time

  • Test multiple antibodies targeting different INTS9 epitopes

• Failed co-immunoprecipitation:

  • Verify protein expression levels of both interaction partners

  • Use tagged versions of proteins for initial studies

  • The INTS9-INTS11 heterodimer requires specific buffer conditions for stability

  • Consider native versus denaturing lysis conditions

How can I address cross-reactivity or nonspecific binding issues with INTS9 antibodies?

To address cross-reactivity concerns:

• Validation experiments:

  • Test antibody on INTS9-depleted samples

  • Perform peptide competition assays with the immunizing peptide

  • Compare results from multiple antibodies recognizing different epitopes

• Application-specific considerations:

  • For Western blot: Include molecular weight markers and verify the expected 75 kDa size

  • For ChIP: Include IgG controls and validate enrichment at known targets

  • For IF/IHC: Include antigen retrieval optimization steps

• Species-specific considerations:

  • Verify sequence homology between your experimental species and the immunogen

  • CST antibody #13945 has confirmed reactivity with human, mouse, rat, and monkey INTS9

  • Some antibodies may be species-limited (e.g., Abcam ab234700 is validated only for human)