ITSN1 Antibody

What is ITSN1 and why is it relevant for research?

ITSN1 (Intersectin 1) is a multifunctional scaffold protein involved in exocytosis, intracellular signal transduction, and actin cytoskeleton reconstruction . The human version has a canonical amino acid length of 1721 residues and a protein mass of 195.4 kilodaltons, with 13 identified isoforms . ITSN1 has gained research significance due to its associations with breast cancer progression, neuroblastoma tumorigenesis, and neurological functions including learning and memory . The protein exists in multiple isoforms, with ITSN1-S (short) and ITSN1-L (long) being the most studied variants, each with distinct cellular functions and localization patterns .

What are the main isoforms of ITSN1 and how do they differ functionally?

ITSN1 exists primarily in two major isoforms:

• ITSN1-S (short isoform): Predominantly expressed in most cell types and tissues. It can localize to both cytoplasm and nucleus in breast cancer cells, with different functions in each compartment . In the cytoplasm, its EH domains interact with PI3KC2α to inhibit cell migration and invasion by inactivating the PI3KC2α-AKT pathway .

• ITSN1-L (long isoform): Particularly important in neuronal tissues. Research using ITSN1-L knockout (ITSN1-LKO) mice demonstrates its critical role in learning, memory formation, and long-term potentiation (LTP) . This isoform appears to be involved in AKT signaling pathways in the hippocampus and influences dendritic spine density on hippocampal pyramidal neurons .

The functional differences between these isoforms make isoform-specific antibodies particularly valuable for targeted research applications.

What types of ITSN1 antibodies are currently available for research?

Current commercially available ITSN1 antibodies include:

• Unconjugated primary antibodies suitable for Western Blot (WB) and ELISA applications

• Antibodies with reactivity to human ITSN1

• Isoform-specific antibodies that can differentiate between ITSN1-S and ITSN1-L

• Antibodies targeting specific domains (SH3, EH, CC domains)

The selection of appropriate ITSN1 antibodies depends on the specific research application, target species, and cellular compartment being studied .

What are the validated methods for detecting ITSN1 expression in tissue samples?

Several validated methods have been employed for ITSN1 detection in tissues:

• RT-PCR analysis: Total RNA extraction using TRizol reagent followed by cDNA synthesis and real-time quantification with SYBR Green PCR kit. ITSN1 expression should be normalized to housekeeping genes like GAPDH and quantified using the 2−∆∆Ct method .

• Western blot analysis: Has been successfully used to detect ITSN1 expression in breast cancer tissues, neuroblastoma cell lines, and brain tissues . This approach can distinguish between ITSN1-S and ITSN1-L isoforms based on molecular weight differences.

• Immunohistochemistry: The Human Protein Atlas provides immunohistochemistry-based expression data for ITSN1 across approximately 20 common cancer types . This technique allows for direct comparison of protein expression between normal and cancerous tissues.

Each method has specific advantages depending on whether you're investigating mRNA expression, protein levels, or cellular localization patterns.

How should ITSN1 antibody validation be performed to ensure specificity?

Proper validation of ITSN1 antibodies is crucial due to the presence of multiple isoforms and domain-specific functions. A comprehensive validation approach should include:

• Western blot analysis with positive and negative controls: Use tissues or cell lines with known ITSN1 expression patterns (e.g., neuroblastoma cell lines like NLF, LAN-1, IMR-5, CHP-134 for positive controls) . For negative controls, consider ITSN1-silenced cell lines generated through shRNA technology as described in neuroblastoma studies .

• Peptide competition assays: Pre-incubate the antibody with purified ITSN1 protein or peptide to confirm signal specificity.

• Cross-reactivity testing: Ensure the antibody doesn't cross-react with closely related proteins, particularly ITSN2.

• Isoform specificity verification: If using isoform-specific antibodies, verify that they correctly distinguish between ITSN1-S (195.4 kDa) and ITSN1-L isoforms .

• Subcellular localization confirmation: Use immunofluorescence with appropriate subcellular markers to confirm the expected localization patterns, noting that ITSN1-S has been found in both cytoplasm and nucleus of breast cancer cells .

What are the optimal conditions for using ITSN1 antibodies in Western blot applications?

Based on published protocols, the following conditions are recommended for optimal Western blot detection of ITSN1:

• Sample preparation: Total protein extraction using standard lysis buffers containing protease inhibitors. Given ITSN1's high molecular weight (195.4 kDa for canonical form), using low percentage (6-8%) SDS-PAGE gels is recommended for proper resolution .

• Transfer conditions: Extended transfer times (overnight at low voltage or 2+ hours at higher voltage) may be necessary for complete transfer of high molecular weight ITSN1 isoforms.

• Blocking and antibody incubation:

  • Blocking: 5% non-fat milk or BSA in TBST for 1-2 hours at room temperature

  • Primary antibody: Typically used at 1:500-1:1000 dilution, incubated overnight at 4°C

  • Secondary antibody: HRP-conjugated at 1:2000-1:5000 dilution for 1-2 hours at room temperature

• Detection: Enhanced chemiluminescence (ECL) systems are commonly used, with exposure times adjusted based on expression levels.

• Controls: Include positive control samples such as neuroblastoma cell lines (NLF, LAN-1, IMR-5) which express high levels of ITSN1 .

How can ITSN1 antibodies be used to investigate its role in breast cancer progression?

ITSN1 antibodies have proven valuable in elucidating this protein's role in breast cancer through several advanced techniques:

• Immunohistochemical analysis: Researchers have used ITSN1 antibodies to compare expression levels between normal breast tissue and carcinoma samples. Studies have found that low ITSN1 expression is significantly associated with advanced clinical cancer stages, suggesting its potential as a prognostic biomarker .

• Subcellular localization studies: ITSN1-S has been discovered in both cytoplasmic and nuclear compartments of breast cancer cells, with different functions in each location. Nuclear ITSN1-S interacts with nuclear DNA helicase II (NDH II) to suppress DNA replication and nascent DNA synthesis by inhibiting R-loops resolution . Antibodies targeting specific domains can help distinguish these compartment-specific interactions.

• Co-immunoprecipitation assays: ITSN1 antibodies enable the investigation of protein-protein interactions, such as the binding between ITSN1-S's EH domains and PI3KC2α, which inhibits cell migration and invasion by inactivating the PI3KC2α-AKT pathway .

• Combined prognostic assessment: Clinical data suggests that combined consideration of both cytoplasmic and nuclear ITSN1-S can serve as an independent prognostic factor in breast cancer patients .

What techniques can be used to study ITSN1's neurological functions using specific antibodies?

ITSN1, particularly the ITSN1-L isoform, plays crucial roles in neurological function. Researchers can employ these techniques with ITSN1 antibodies:

• Immunohistochemistry of brain tissue sections: To examine expression patterns in specific neuronal populations and dendritic spines. Studies using ITSN1-L knockout mice have demonstrated its importance in dendritic spine formation and maintenance .

• Co-localization studies: Using confocal microscopy with ITSN1 antibodies alongside synaptic markers to investigate its role in synaptic function and plasticity.

• Biochemical fractionation of brain tissue: Combined with Western blotting to determine ITSN1 distribution in different neuronal compartments (synaptic vesicles, post-synaptic density, etc.).

• Proximity ligation assays: To investigate protein-protein interactions in intact neurons, particularly how ITSN1 interacts with components of the synaptic vesicle recycling machinery.

• Long-term potentiation (LTP) studies: Combined with ITSN1 antibody intervention to assess the protein's role in synaptic plasticity. Research has shown that ITSN1-LKO mice exhibit impaired LTP and deficits in learning and long-term spatial memory .

How can researchers quantitatively assess ITSN1 expression in tumor samples?

For quantitative assessment of ITSN1 in tumor samples, researchers should consider these methodological approaches:

• RT-qPCR standardization:

  • Use validated reference genes like GAPDH for normalization

  • Apply the 2−∆∆Ct method for relative quantification

  • Consider using absolute quantification with standard curves for more precise measurements

• Western blot densitometry:

  • Normalize ITSN1 band intensity to loading controls (β-actin, GAPDH)

  • Use digital image analysis software (ImageJ, etc.) for quantification

  • Include calibration standards on each blot for cross-blot comparisons

• Immunohistochemical scoring systems:

  • Implement standardized scoring systems like H-score (0-300) or Allred score (0-8)

  • Consider automated digital pathology platforms for more objective quantification

  • Establish thresholds for "high" versus "low" expression (e.g., relative ITSN1 expression >1.3 as high expression)

• Statistical considerations:

  • Analyze correlations between ITSN1 expression and clinicopathological characteristics using χ² test

  • Construct survival curves using Kaplan-Meier method and analyze by log-rank test

  • Consider ITSN1 expression in multivariate models along with established prognostic factors

What are common challenges when detecting ITSN1 in experimental samples and how can they be overcome?

Researchers frequently encounter these challenges when working with ITSN1 antibodies:

• High molecular weight detection issues:

  • Problem: Incomplete transfer of high molecular weight ITSN1 (195.4 kDa) during Western blotting

  • Solution: Use lower percentage gels (6-8%), extend transfer time, or implement semi-dry transfer systems designed for high molecular weight proteins

• Isoform distinction difficulties:

  • Problem: Inability to distinguish between ITSN1-S and ITSN1-L isoforms

  • Solution: Select isoform-specific antibodies or optimize gel conditions to clearly separate these variants based on molecular weight differences

• Low signal intensity:

  • Problem: Weak ITSN1 signal, particularly in tissues with naturally low expression

  • Solution: Implement signal enhancement methods such as tyramide signal amplification, increase protein loading, or optimize antibody concentrations and incubation conditions

• Non-specific binding:

  • Problem: Multiple bands or background signal

  • Solution: Increase blocking stringency, optimize antibody dilutions, or use monoclonal antibodies with higher specificity

• Subcellular localization assessment:

  • Problem: Difficulty confirming nuclear versus cytoplasmic localization of ITSN1-S

  • Solution: Perform subcellular fractionation followed by Western blotting, or use super-resolution microscopy with co-localization markers for nuclear and cytoplasmic compartments

How should researchers interpret conflicting data regarding ITSN1 expression patterns?

When faced with conflicting ITSN1 expression data, consider these methodological approaches:

• Isoform-specific analysis: Verify whether discrepancies might be due to differential expression of specific ITSN1 isoforms. The literature indicates that ITSN1-S is the predominant isoform in many cell types, but ITSN1-L can be significantly expressed in specific contexts like certain neuroblastoma cell lines (LAN2, LAN5, and IMR-32) .

• Tissue/cell type considerations: Expression patterns may legitimately differ between tissue types. For example, ITSN1 shows variable expression across different neuroblastoma cell lines, with notable differences between MYCN-amplified and non-amplified cells .

• Methodological evaluation: Assess whether conflicting results stem from different detection methods. RT-PCR might show different patterns than protein-based detection methods due to post-transcriptional regulation.

• Antibody validation assessment: Verify that all antibodies used across different studies have been properly validated and target the same epitopes. Antibodies recognizing different domains of ITSN1 may yield different results.

• Cellular stress and experimental conditions: ITSN1 expression and localization can be affected by cellular stress conditions. Standardize experimental conditions when comparing across studies .

What controls are essential when studying ITSN1 knockdown or overexpression effects?

When manipulating ITSN1 expression levels, these controls are essential for result validation:

• Knockdown controls:

  • Include multiple shRNA or siRNA constructs targeting different regions of ITSN1 to rule out off-target effects (e.g., sh#1 and sh#2 as demonstrated in neuroblastoma studies)

  • Implement scrambled shRNA controls (pSCR) and empty vector controls (pSR) as demonstrated in published protocols

  • Quantify knockdown efficiency at both mRNA (RT-qPCR) and protein (Western blot) levels

  • Monitor cell viability and proliferation rates to ensure observed phenotypes aren't due to general cytotoxicity

• Overexpression controls:

  • Include empty vector transfection controls

  • Verify expression levels of both endogenous and exogenous ITSN1

  • Implement rescue experiments to confirm specificity (e.g., overexpression of PI3K-C2β rescued the soft agar growth of ITSN1-silenced cells)

  • Assess potential artifacts from protein tagging by comparing tagged and untagged constructs

• Functional validation:

  • Assess expected ITSN1 functions, such as endocytosis (transferrin internalization)

  • Monitor established downstream targets and pathways (e.g., PI3K-C2β-AKT pathway)

  • Perform functional assays relevant to the disease/process being studied (e.g., soft agar growth for tumorigenesis studies)

How are ITSN1 antibodies being used to investigate novel cellular functions?

Recent research has expanded our understanding of ITSN1's cellular functions beyond its traditional roles in endocytosis:

• Nuclear functions exploration: Newly developed antibodies capable of distinguishing nuclear ITSN1-S have revealed its unexpected role in suppressing DNA replication through interaction with nuclear DNA helicase II. This research direction is uncovering how endocytic proteins can have distinct nuclear functions .

• Tumorigenesis mechanisms: Antibody-based studies have established that ITSN1 is required for neuroblastoma tumorigenesis through the ITSN1-PI3K-C2β pathway, representing the first demonstration of this pathway's requisite role in human cancer .

• Compartment-specific signaling: Advanced immunofluorescence techniques combined with subcellular fractionation are revealing how ITSN1 coordinates different signaling pathways in distinct cellular compartments. For example, the interaction between cytoplasmic ITSN1-S's EH domains and PI3KC2α inhibits cell migration, while nuclear ITSN1-S interacts with NDH II to suppress DNA replication .

• Long non-coding RNA interactions: Recent studies have identified correlations between lnc-ITSN1-2 and inflammatory disorders like IBD, suggesting complex regulatory relationships between ITSN1, its genetic locus, and disease mechanisms .

What emerging techniques are enhancing ITSN1 antibody-based research?

Several cutting-edge techniques are advancing ITSN1 antibody applications:

• Proximity labeling approaches: BioID and APEX2-based techniques coupled with ITSN1 antibodies are enabling the identification of transient or weak interaction partners in specific cellular compartments.

• Live-cell imaging with nanobodies: Development of ITSN1-specific nanobodies is facilitating real-time visualization of ITSN1 dynamics in living cells with minimal interference with function.

• Super-resolution microscopy: Techniques like STORM and PALM combined with highly specific ITSN1 antibodies are revealing previously undetectable details about ITSN1's subcellular organization and interaction networks.

• Mass spectrometry-based interactomics: IP-MS approaches using ITSN1 antibodies are identifying comprehensive interaction networks in different cellular contexts and disease states.

• Single-cell analysis: Integration of ITSN1 antibodies with single-cell technologies is revealing cell-to-cell variability in expression and function within heterogeneous tissues.

How can researchers integrate ITSN1 antibody studies with broader -omics approaches?

Integration of ITSN1 antibody research with multi-omics approaches offers powerful insights:

• Proteogenomic integration:

  • Correlate ITSN1 protein levels (detected by antibodies) with genomic/transcriptomic data

  • Identify potential post-transcriptional regulation mechanisms explaining discrepancies between mRNA and protein levels

  • Investigate how genetic alterations affect ITSN1 expression, localization, and function

• Phosphoproteomics coupling:

  • Use phospho-specific ITSN1 antibodies alongside global phosphoproteomics

  • Map how ITSN1 phosphorylation states correlate with activation of specific signaling pathways

  • Identify kinases responsible for ITSN1 regulation in different cellular contexts

• Spatial transcriptomics integration:

  • Combine ITSN1 immunohistochemistry with spatial transcriptomics data

  • Map correlation between ITSN1 protein expression and local gene expression programs

  • Identify tissue microenvironment factors influencing ITSN1 expression patterns

• Network analysis approaches:

  • Position ITSN1 within protein-protein interaction networks using antibody-based interactome data

  • Identify hub proteins and pathways connected to ITSN1 in health and disease contexts

  • Apply machine learning to predict functional consequences of ITSN1 dysregulation