telo2 Antibody

What is TELO2 and what cellular functions does it regulate?

TELO2 (Telomere Maintenance 2 Homolog) is a multifunctional protein that plays critical roles in several cellular processes. It functions as:

• A regulator of the DNA damage response (DDR)

• Part of the TTT complex (TELO2, TTI1, TTI2) that stabilizes phosphatidylinositol 3-kinase-related protein kinase (PIKK) family proteins

• A component involved in the assembly and maintenance of mTORC1 and mTORC2 complexes

• An S-phase checkpoint protein in the cell cycle

• A potential regulator of telomere length

Specifically, TELO2 promotes assembly, stabilizes and maintains the activity of mTORC1 and mTORC2 complexes, which regulate cell growth and survival in response to nutrient and hormonal signals. Research has shown that TELO2 interacts with RICTOR, a component of mTORC2, to influence cancer progression .

Where is TELO2 protein localized in human cells?

The subcellular localization of TELO2 varies by cell type and can be detected using immunofluorescence and immunohistochemistry:

These findings indicate that TELO2 may have different subcellular distributions depending on cell type, suggesting context-dependent functions.

What is the molecular weight of TELO2 protein?

TELO2 is a 837 amino acid protein with a calculated molecular weight of 92 kDa, which matches the observed molecular weight in Western blot analyses . When running Western blot experiments with TELO2 antibodies, researchers should expect to detect a band at approximately 92 kDa.

How does TELO2 function in cancer biology and what are its context-dependent roles?

TELO2 exhibits complex, context-dependent functions in cancer biology:

Colorectal Cancer (CRC):

• TELO2 functions as a "double-edged sword" in CRC progression

• Under normal growth conditions with serum supplementation:

  • TELO2 binds with RICTOR as part of the mTORC2 complex

  • This promotes proliferation, migration, and invasion through the AKT pathway

  • Downregulation of TELO2 inhibits malignant biological behavior of CRC cells

• Under serum deprivation (mimicking heavy tumor burden conditions):

  • TELO2 is ubiquitinated by RICTOR through an mTORC2-independent manner

  • The stability of TELO2 is lower in serum-deprived cells

  • RICTOR knockdown prolongs the half-life of TELO2 under serum deprivation

High-Grade Gliomas:

• Overexpression of TELO2 mRNA correlates with shorter survival outcomes

• TELO2 mRNA expression is significantly higher in human glioma cell lines (LN229, GBM8401, U118MG) compared to normal brain tissue

• TELO2 may serve as a prognostic marker and therapeutic target in high-grade gliomas

This dual role suggests that TELO2-targeted therapies would need to consider the microenvironmental context of the tumor.

What is the relationship between TELO2 and the mTOR signaling pathway?

TELO2 has intricate connections with the mTOR signaling pathway:

• Complex Formation:

  • TELO2 promotes assembly, stabilizes, and maintains the activity of both mTORC1 and mTORC2 complexes

  • Part of the TTT complex that is required for PIKK family protein stability, including mTOR

• Differential Effects on mTORC1 vs mTORC2:

  • Inhibition of TELO2 decreases mTOR expression without changing RICTOR (mTORC2) and RAPTOR (mTORC1) levels

  • Affects downstream mTORC2 signaling by decreasing phosphorylated Akt Ser473 levels

  • Has more subtle effects on mTORC1 targets (p-S6K1 and p-4EBP1)

• Reciprocal Regulation:

  • RICTOR (mTORC2 component) can regulate TELO2 stability through ubiquitination under serum deprivation

  • RICTOR knockdown inhibits TELO2 slightly under normal conditions

  • Under serum deprivation, RICTOR knockdown paradoxically increases TELO2 expression

• Binding Dynamics:

  • Binding between RICTOR and TELO2 increases in serum-deprived cells

  • This binding occurs in the absence of mTOR, suggesting an mTORC2-independent interaction

These findings reveal a complex interdependence between TELO2 and mTOR signaling that is modulated by cellular nutrient status.

How does TELO2 contribute to DNA damage response and genomic stability?

While the search results provide limited direct information on this topic, TELO2's role in DNA damage response (DDR) can be inferred:

• TTT Complex Formation:

  • TELO2 forms the TTT complex with TTI1 and TTI2

  • This complex is required to stabilize protein levels of the phosphatidylinositol 3-kinase-related protein kinase (PIKK) family proteins

  • PIKK family includes key DDR proteins like ATM, ATR, and DNA-PKcs

• Stress Response:

  • The TTT complex is involved in cellular resistance to various DNA damage stresses:

    • Ionizing radiation (IR)

    • Ultraviolet (UV) radiation

    • Mitomycin C (MMC)

• Protein Folding:

  • Together with the TTT complex and HSP90, TELO2 may participate in the proper folding of newly synthesized PIKKs

  • This ensures these critical DDR kinases are functionally active

• S-phase Checkpoint:

  • TELO2 functions as an S-phase checkpoint protein in the cell cycle

  • This likely contributes to preventing replication of damaged DNA

These functions collectively suggest that TELO2 helps maintain genomic stability by ensuring proper DDR signaling and checkpoint activation in response to DNA damage.

What are the optimal conditions for using TELO2 antibodies in Western blot applications?

Based on the available data, here are the recommended conditions for Western blot applications with TELO2 antibodies:

General recommendations for optimal Western blot results:

• Use 30 μg of whole protein lysates for detection

• Use 4-12% NuPAGE Bis-Tris gel for electrophoresis

• For investigating interactions with mTOR complexes:

  • Use either 1% Triton (strong lysis buffer which can depolymerize the mTOR complex)

  • Or CHAPS (mild buffer which can maintain the integrated mTOR complex)

• For protein stability studies, consider conducting cycloheximide (CHX) chase experiments

What are the key considerations for immunohistochemistry and immunofluorescence applications with TELO2 antibodies?

For successful immunohistochemistry (IHC) and immunofluorescence (IF) experiments with TELO2 antibodies:

Immunohistochemistry (IHC):

Immunofluorescence (IF):

General recommendations:

• Always perform optimization experiments to determine the ideal antibody concentration for your specific tissue/cell type

• Include appropriate positive controls (e.g., human urinary bladder cancer tissue is known to stain positive for TELO2)

• For IHC, consider testing both TE buffer pH 9.0 and citrate buffer pH 6.0 for antigen retrieval

• The optimal working dilution should be determined by the end user based on the specific experimental conditions

How can TELO2 antibodies be used to study protein-protein interactions in the mTOR pathway?

Based on the research findings, here are recommended methodologies for studying TELO2 interactions with mTOR pathway components:

Co-Immunoprecipitation (Co-IP):

• Buffer Selection is Critical:

  • For studying intact mTOR complexes: Use CHAPS buffer (mild buffer that maintains integrated mTOR complex)

  • For studying individual component interactions: Use 1% Triton (strong lysis buffer that can depolymerize mTOR complex)

• Protocol Overview:

  • Pre-incubate lysates with protein G PLUS-Agarose beads

  • Incubate equal amounts of protein (approximately 500 μg) with antibodies against TELO2 or RICTOR (1 μg)

  • Wash beads with appropriate buffer (1% Triton or CHAPS) at 4°C, 200 × g, three times

  • Load 1% of the input to detect protein levels

• Detecting Specific Interactions:

  • The TELO2-RICTOR interaction is increased in serum-deprived cells

  • Under serum deprivation, this interaction occurs independently of mTOR

Ubiquitination Assays:

• To study RICTOR-mediated ubiquitination of TELO2:

  • Transfect cells with His-ubiquitin and/or RICTOR siRNA

  • Culture cells with or without serum

  • Perform ubiquitination assays using standard protocols

Protein Stability Assays:

• For cycloheximide (CHX) chase experiments:

  • Treat cells with CHX to inhibit new protein synthesis

  • Harvest cells at various time points

  • Analyze TELO2 protein levels by Western blot

  • Note that TELO2 stability is higher in cells cultured with serum compared to serum-deprived conditions

  • RICTOR knockdown prolongs TELO2 half-life under serum deprivation

Bioinformatics Prediction:

• STRING database (https://string-db.org) can be used for predicting protein combinations before experimental validation

These methodologies can help researchers elucidate the complex interactions between TELO2 and components of the mTOR pathway under different cellular conditions.

What are the best experimental approaches to study TELO2's role in cancer progression?

Based on published research, the following experimental approaches are recommended for investigating TELO2's role in cancer:

In Vitro Studies:

• Gene Knockdown and Overexpression:

  • Use TELO2 shRNA (e.g., sc-93308-SH from Santa Cruz) for stable knockdown

  • Use pLPC-Myc-TELO2 (Addgene #22802) for overexpression

  • Generate stable cell lines with puromycin selection

• Proliferation and Colony Formation Assays:

  • WST-1 assay for cell proliferation

  • Soft agar assay for anchorage-independent growth

• Cell Cycle Analysis:

  • Flow cytometry to assess cell cycle distribution

  • Evaluate G1/S phase checkpoint function

• Migration and Invasion Assays:

  • Wound healing assay for cell migration assessment

    • Seed cells until >95% confluency, wound with pipette tip

    • Capture images at 24 and 48h post-wounding

  • Matrigel Invasion Chamber (BD Biosciences) for invasion assessment

    • Place 1×10^5 cells in serum-free media on Transwell membrane

    • Fill lower chamber with complete medium

    • After 24h, stain with 0.005% crystal violet and count under microscope

• Protein Interaction and Regulation Studies:

  • Co-IP to examine TELO2 interactions with pathway components

  • CHX chase assays to determine protein stability

  • Ubiquitination assays to study post-translational regulation

Expression Analysis in Patient Samples:

• Tissue Microarray Analysis:

  • IHC staining using validated TELO2 antibodies (dilution 1:50-1:200)

  • Compare expression between cancer tissue and adjacent normal tissue

  • Use two-independent non-parametric test (Mann-Whitney U test)

• Survival Analysis:

  • Kaplan-Meier and log rank tests to analyze survival differences

  • Correlate TELO2 expression with patient outcomes

• Correlation with Clinical Features:

  • Chi-square test to analyze relationships between TELO2 expression and clinical features

  • Spearman's correlation for quantifying relationships between TELO2 and other proteins (e.g., RICTOR)

Bioinformatics Approaches:

• Gene Expression Database Analysis:

  • Use GEO profiles to analyze TELO2 expression across cancer types

  • GENT2 database to examine expression in cancer cell lines

  • Apply conditional inference tree via 'party' package with R language (R 3.1.2 software) to determine cut-off values

• Protein Interaction Networks:

  • STRING database for protein-protein interaction prediction

  • Visualize networks to identify potential new interactors