TOM1L1 Antibody

What is TOM1L1 and what are its key functional domains?

TOM1L1 (Target of Myb1-like 1) is an adaptor protein of the Tom1 family that functions as both a substrate and activator of Src family protein-tyrosine kinases (SFK). It contains several key domains including VHS, GAT, linker region, and a C-terminal domain. The C-terminal region contains a 457EEI sequence that creates a perfect phosphorylation site for Src and binding to its SH2 domain, plus a RLP421PLP motif with potential high affinity for SrcSH3. TOM1L1 also contains binding sites for signaling proteins like Grb2 (Tyr441) and the p85 subunit of phosphatidylinositol 3-kinase (Tyr392), though Tyr457 is the major Src phosphorylation site in vivo . The linker region between the GAT domain and C-terminus (amino acids 291 to 387) has been identified as functionally important for TOM1L1's biological activity .

What is the predicted molecular weight of TOM1L1 versus observed weight in experimental applications?

While the calculated molecular weight of TOM1L1 is approximately 39 kDa (comprising 346 amino acids) , researchers frequently observe a higher apparent molecular weight of approximately 53 kDa in Western blot applications . This discrepancy between predicted and observed molecular weights is likely due to post-translational modifications, particularly phosphorylation events that are known to occur on TOM1L1. Additionally, some researchers have noted a 40 kDa protein that is frequently observed when using TOM1L1 antibodies , which may represent an isoform or processed form of the protein.

What species reactivity is typically observed with commercial TOM1L1 antibodies?

Most commercially available TOM1L1 antibodies show reactivity with human, mouse, and rat samples . This cross-reactivity reflects the conservation of the TOM1L1 protein across mammalian species. When selecting antibodies for particular experimental systems, it's advisable to confirm specific species reactivity with the manufacturer. Some antibodies may have more limited species reactivity, such as those specifically validated only for human samples .

What are the validated applications for TOM1L1 antibodies in molecular and cellular research?

TOM1L1 antibodies have been successfully employed in multiple experimental applications:

When designing experiments, researchers should validate the antibody in their specific experimental system, as performance can vary depending on sample type, preparation method, and detection system used.

How can researchers optimize Western blot protocols for TOM1L1 detection?

For optimal Western blot detection of TOM1L1:

• Sample preparation: Include phosphatase inhibitors in lysis buffers to preserve phosphorylation states, particularly when studying TOM1L1 regulation by Src kinases or ERBB2 signaling .

• Gel percentage: Use 8-10% polyacrylamide gels to achieve good separation around the 40-55 kDa range where TOM1L1 migrates.

• Transfer conditions: Optimize transfer time for proteins in the 40-55 kDa range (typically 1-1.5 hours at 100V or overnight at 30V).

• Blocking: Use 5% BSA in TBST rather than milk when detecting phosphorylated forms of TOM1L1, as milk contains phosphoproteins that may increase background.

• Antibody dilution: Start with a 1:1000 dilution for Western blot and adjust based on signal intensity and background .

• Controls: Include positive controls such as HeLa cell lysates, which have been validated for TOM1L1 expression . For phospho-specific studies, include samples treated with phosphatase to confirm specificity.

What methods are recommended for validating TOM1L1 antibody specificity?

To confirm the specificity of TOM1L1 antibodies:

• Preblocking experiments: Incubate the antibody with its cognate antigen before immunostaining, which should abolish specific signal. This approach has been demonstrated effective in prior studies, where TOM1L1 was not detected by antibody preblocked by its cognate antigen .

• siRNA knockdown: Transfect cells with TOM1L1-specific siRNA and confirm reduction in signal by Western blot or immunostaining.

• Overexpression studies: Compare signal between cells overexpressing TOM1L1 versus control cells.

• Peptide competition assays: Determine specificity by competing the antibody binding with increasing concentrations of the immunizing peptide.

• Multiple antibody validation: Use different antibodies recognizing distinct epitopes of TOM1L1 to confirm the observed patterns.

How does TOM1L1 contribute to ERBB2-positive breast cancer progression, and how can researchers study this relationship?

TOM1L1 is co-amplified with ERBB2 and defines a subgroup of HER2+/ER+ tumors with early metastatic relapse . To study this relationship:

• Analyze co-expression: Researchers can use dual immunostaining approaches to evaluate co-expression patterns of TOM1L1 and ERBB2 in breast cancer tissues.

• Investigate invasion mechanisms: TOM1L1 enhances invasiveness of ERBB2-transformed cells through membrane-bound MT1-MMP-dependent activation of invadopodia . This can be studied using:

  • Invadopodia formation assays with fluorescent gelatin degradation

  • 3D invasion assays in matrigel or collagen matrices

  • Live-cell imaging with fluorescently tagged TOM1L1 and MT1-MMP

• Examine phosphorylation status: ERBB2 signaling leads to phosphorylation of TOM1L1 on Ser321, which promotes GAT-dependent association with TOLLIP and trafficking of MT1-MMP . Researchers can:

  • Use phospho-specific antibodies against Ser321

  • Employ phospho-mimetic (S321D) and phospho-dead (S321A) TOM1L1 mutants

  • Perform co-immunoprecipitation to track TOM1L1-TOLLIP interactions

• Correlation with clinical outcomes: Analyze TOM1L1 expression in patient samples and correlate with metastatic progression and survival in ERBB2-positive breast cancers.

How can researchers differentiate between TOM1L1's SRC-dependent and SRC-independent functions in experimental systems?

TOM1L1 exhibits both SRC-dependent and SRC-independent functions that can be studied through different experimental approaches:

• For SRC-dependent functions:

  • Use SRC inhibitors like PP2 or dasatinib to block SRC-mediated phosphorylation of TOM1L1

  • Employ TOM1L1 mutants where Y457 is mutated to phenylalanine (Y457F) to prevent SRC phosphorylation

  • Conduct in vitro kinase assays with purified SRC and TOM1L1 to measure direct phosphorylation

  • Assess co-immunoprecipitation of TOM1L1 with SRC under various conditions

• For SRC-independent functions (e.g., in ERBB2-driven invasion):

  • Use ERBB2 inhibitors (lapatinib, trastuzumab) to block ERBB2-mediated effects while maintaining SRC activity

  • Employ TOM1L1 mutations in the GAT domain to disrupt TOLLIP interaction while preserving SRC binding

  • Perform RNAi-mediated knockdown of TOLLIP to specifically inhibit the TOM1L1-TOLLIP-MT1-MMP trafficking pathway

  • Use MT1-MMP inhibitors to block downstream effects while maintaining TOM1L1-TOLLIP interactions

• Comparative analysis:

  • Monitor cellular readouts (proliferation, invasion) in parallel experiments with SRC versus ERBB2 inhibition

  • Perform proteomics analysis of TOM1L1 interactors under conditions of SRC inhibition versus ERBB2 inhibition

How can researchers investigate the regulatory mechanisms controlling TOM1L1 phosphorylation beyond Src-mediated events?

While Src-mediated phosphorylation of TOM1L1 at Y457 is well-documented , ERBB2 signaling leads to phosphorylation at Ser321 . Researchers can explore additional regulatory mechanisms through:

• Phosphoproteomic analysis:

  • Use mass spectrometry-based approaches to identify all phosphorylation sites on TOM1L1

  • Compare phosphorylation patterns after treatment with various growth factors or kinase inhibitors

  • Employ SILAC or TMT labeling for quantitative analysis of dynamic phosphorylation

• Kinase prediction and validation:

  • Use in silico tools (NetPhos, GPS, etc.) to predict potential kinases for identified phosphorylation sites

  • Conduct in vitro kinase assays with purified kinases and TOM1L1

  • Employ specific kinase inhibitors to validate predictions in cellular models

• Mutagenesis studies:

  • Generate phospho-dead and phospho-mimetic mutations at identified sites

  • Assess functional consequences through trafficking assays, protein-protein interaction studies, and cellular phenotypes

  • Use domain-specific mutants to determine functional relationships between phosphorylation and protein domains

• Temporal dynamics:

  • Employ real-time imaging with phospho-specific biosensors to track TOM1L1 phosphorylation kinetics

  • Analyze temporal relationships between receptor activation, TOM1L1 phosphorylation, and trafficking events

What experimental approaches can resolve the apparent contradiction between TOM1L1's negative regulation of SFK signaling and its pro-tumorigenic role in ERBB2-positive breast cancer?

The literature reveals an apparent contradiction: TOM1L1 negatively regulates SFK mitogenic signaling yet promotes invasiveness in ERBB2-positive breast cancer . Researchers can address this paradox through:

• Context-dependent analysis:

  • Compare TOM1L1 function in PDGF versus ERBB2 signaling contexts

  • Analyze differential protein complexes formed by TOM1L1 in each context

  • Investigate cell type-specific effects (fibroblasts versus epithelial cells)

• Domain-specific functions:

  • Use domain-specific mutations to separate TOM1L1's roles in SFK regulation versus MT1-MMP trafficking

  • Analyze the linker region (amino acids 291-387) which may have distinct functions in different contexts

  • Employ domain-swapping experiments between TOM1L1 and other Tom1 family members

• Signaling pathway integration:

  • Map how TOM1L1 differentially affects MAPK, PI3K, and other downstream pathways in different contexts

  • Use phosphoproteomic analysis to identify differential effects on the global phosphoproteome

  • Perform pathway inhibition studies to determine which downstream pathways are critical for each function

• Temporal dynamics and localization:

  • Track TOM1L1 subcellular localization during PDGF versus ERBB2 signaling

  • Analyze temporal differences in TOM1L1 recruitment, phosphorylation, and function

  • Use optogenetic approaches to control TOM1L1 localization and analyze context-dependent functions

What methodologies can researchers employ to study TOM1L1's role in trafficking MT1-MMP to invadopodia?

TOM1L1 promotes trafficking of MT1-MMP from endocytic compartments to invadopodia . Researchers can study this mechanism through:

• Live-cell imaging approaches:

  • Use dual-color confocal microscopy with fluorescently tagged TOM1L1 and MT1-MMP

  • Employ TIRF microscopy to visualize events at the plasma membrane

  • Implement photoactivatable or photoconvertible MT1-MMP to track specific pools of the protease

• Endosomal tracking:

  • Use endosomal markers (Rab5, Rab7, Rab11) to identify specific compartments involved in MT1-MMP trafficking

  • Perform live-cell imaging of TOM1L1, TOLLIP, and MT1-MMP with endosomal markers

  • Employ structured illumination or super-resolution microscopy for detailed visualization

• Functional invadopodia assays:

  • Use fluorescent gelatin degradation assays to measure invadopodia activity

  • Correlate gelatin degradation with TOM1L1 and MT1-MMP localization

  • Implement TOM1L1 mutations (particularly S321 phospho-mutants) and assess effects on invadopodia formation and activity

• Biochemical approaches:

  • Isolate invadopodia-enriched fractions and analyze TOM1L1 and MT1-MMP content

  • Perform proximity labeling (BioID, APEX) with TOM1L1 as bait to identify invadopodia-specific interactors

  • Use co-immunoprecipitation to track TOM1L1-TOLLIP-MT1-MMP complexes under various conditions

How can researchers address inconsistent molecular weight observations in Western blot analysis of TOM1L1?

Researchers may observe TOM1L1 at different molecular weights ranging from 39-53 kDa . To address this variability:

• Sample preparation considerations:

  • Ensure complete protein denaturation with sufficient SDS and heating

  • Include phosphatase inhibitors to preserve phosphorylation states

  • Use freshly prepared samples when possible, as degradation may generate lower molecular weight bands

• Resolution optimization:

  • Use gradient gels (4-15%) to better resolve potential isoforms

  • Extend running time to improve separation in the 30-60 kDa range

  • Consider using Phos-tag acrylamide gels to separate phosphorylated forms

• Analytical approaches:

  • Perform immunoprecipitation followed by mass spectrometry to identify the exact nature of different bands

  • Use phosphatase treatment to determine if higher molecular weight forms are due to phosphorylation

  • Compare antibodies targeting different epitopes to help identify specific isoforms

• Controls and validation:

  • Include recombinant TOM1L1 protein as a molecular weight reference

  • Use lysates from cells overexpressing TOM1L1 alongside endogenous samples

  • Consider TOM1L1 knockdown samples to confirm specificity of all observed bands

What strategies can resolve non-specific binding issues with TOM1L1 antibodies in immunohistochemistry applications?

When performing immunohistochemistry with TOM1L1 antibodies, researchers may encounter non-specific binding. To improve specificity:

• Antibody validation:

  • Perform antibody preblocking with cognate antigen, which should eliminate specific staining

  • Include positive controls (tissues known to express TOM1L1, such as brain) and negative controls

  • Compare staining patterns with multiple antibodies targeting different TOM1L1 epitopes

• Protocol optimization:

  • Test multiple antigen retrieval methods (citrate buffer pH 6.0, EDTA buffer pH 9.0)

  • Optimize antibody concentration through titration experiments (starting from 1:20-1:200)

  • Extend blocking time and use more stringent blocking reagents (add 0.1-0.3% Triton X-100 to blocking buffer)

• Detection system considerations:

  • Compare different detection systems (HRP-polymer, biotinylated secondary antibodies)

  • If using fluorescent detection, employ spectral unmixing to separate specific signal from autofluorescence

  • Consider amplification methods for weak signals (tyramide signal amplification)

• Advanced approaches:

  • Implement multiplex immunostaining to correlate TOM1L1 with known markers

  • Use automated staining platforms to ensure consistency

  • Consider RNAscope or BaseScope in situ hybridization as complementary approaches to validate protein expression patterns

How might researchers explore the potential of TOM1L1 as a prognostic biomarker in ERBB2-positive breast cancers?

Given that TOM1L1 is co-amplified with ERBB2 and defines a subgroup of HER2+/ER+ tumors with early metastatic relapse , researchers could:

What experimental systems would be most appropriate for studying TOM1L1's role in cancer metastasis?

To investigate TOM1L1's contribution to metastatic processes, researchers could employ:

• In vitro 3D models:

  • Organoid cultures from primary tumors

  • Spheroid invasion assays in collagen or matrigel

  • Microfluidic devices to study invasion through defined matrices

  • Co-culture systems with stromal and immune components

• In vivo metastasis models:

  • Orthotopic xenograft models with ERBB2+ breast cancer cells

  • Patient-derived xenografts from ERBB2+ tumors

  • Genetic mouse models with conditional TOM1L1 expression

  • Intravital imaging to track invadopodia formation and metastatic spread

• Systems for mechanistic studies:

  • CRISPR/Cas9-engineered cell lines with TOM1L1 mutations

  • Inducible TOM1L1 expression systems to study temporal effects

  • Domain-specific knock-in mutations to dissect functional regions

  • Dual recombinase systems for tissue-specific manipulation

• Translational approaches:

  • Ex vivo culture of circulating tumor cells from patients

  • "Metastasis-on-a-chip" microfluidic devices

  • Correlation of TOM1L1 expression with circulating tumor DNA and other liquid biopsy parameters

  • Integration with immune infiltration data to explore relationships with immunosurveillance