ttyh3 Antibody

What is TTYH3 and what are its structural characteristics?

TTYH3 (Tweety homolog 3) is a member of the Tweety protein family that functions as a suspected large-conductance Ca(2+)-activated chloride channel. In humans, the canonical protein has a reported length of 523 amino acid residues and a mass of 57.5 kDa . The protein is primarily localized in the cell membrane and is expressed in excitable tissues .

How many TTYH3 isoforms exist and what are their functional differences?

Up to four different isoforms have been reported for the TTYH3 protein . These isoforms result from alternative splicing and may have distinct functional properties. The specific functional differences between these isoforms remain an active area of investigation, as research on isoform-specific roles is still developing. When designing experiments involving TTYH3, researchers should consider which isoform(s) they are targeting and select antibodies that can differentiate between them if isoform-specific analysis is required.

What are the evolutionary implications of the Tweety gene family?

The tweety genes encode gated chloride channels found across diverse taxonomic groups including animals, plants, and simple eukaryotes, indicating their deep evolutionary origin . TTYH3 gene orthologs have been reported in multiple species including mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken . This conservation across species suggests fundamental biological functions and makes comparative studies valuable for understanding TTYH3 biology.

What are the primary applications of TTYH3 antibodies in research?

TTYH3 antibodies are primarily used for the immunodetection of the Tweety family member 3 protein. Common experimental applications include:

• Western Blot analysis - The most common application for detecting and quantifying TTYH3 protein expression

• Immunohistochemistry - For examining TTYH3 expression patterns in tissue samples

• Immunoprecipitation - For studying protein-protein interactions involving TTYH3

• Flow cytometry - For analyzing TTYH3 expression at the cellular level

When selecting a TTYH3 antibody, researchers should consider the specific application, species reactivity, and the epitope recognized by the antibody, as these factors significantly impact experimental outcomes.

What methodological approaches are most effective for studying TTYH3 function?

Based on published research, effective approaches for studying TTYH3 function include:

• RNA interference (RNAi) - Lentiviral-mediated knockdown of TTYH3 has been successfully used to study its role in cancer cell lines

• Protein expression analysis - Western blotting to quantify TTYH3 protein levels and correlate with phenotypic changes

• Cell proliferation assays - Cell counting over multiple days to evaluate growth effects of TTYH3 manipulation

• Sphere formation assays - For assessing cancer stem cell-like properties

• Migration and invasion assays - Including wound closure and transwell migration assays to evaluate metastatic potential

• Bioinformatic analyses - Using databases like TCGA, UALCAN, GEPIA2, and Oncomine to correlate expression with clinical outcomes

These approaches provide complementary insights into TTYH3 function in different biological contexts.

How does TTYH3 expression correlate with cancer progression?

TTYH3 expression has been found to be elevated in multiple cancer types compared to normal tissues. Specifically:

• In bladder urothelial carcinoma (BLCA), mRNA expression of TTYH3 is significantly higher in cancer tissues than in normal tissues, as demonstrated using TCGA dataset analysis through UALCAN and GEPIA2 web tools

• In gastric cancer, elevated TTYH3 expression correlates with poor patient outcomes

• In ovarian cancer (OC), increased TTYH3 expression predicts unfavorable prognosis, particularly in patients with serous, late-stage, and poorly differentiated tumors

The following table summarizes TTYH3's prognostic significance in ovarian cancer across different clinical stages:

*P<0.05

What molecular mechanisms underlie TTYH3's role in cancer progression?

Research suggests TTYH3 promotes cancer progression through several mechanisms:

• MAPK signaling pathway modulation - In TTYH3 knockdown bladder cancer cell lines, protein levels of H-Ras, A-Raf, phospho-MEK, and phospho-ERK decreased, indicating TTYH3 may enhance MAPK pathway activation

• Regulation of transcription factors - TTYH3 knockdown resulted in decreased expression of c-Fos and c-Jun, which are critical regulators of cell migration and invasion

• Interaction with growth factor receptors - Levels of phosphorylated FGFR1 were decreased in TTYH3 knockdown cell lines

• Potential interactions with multiple signaling proteins - Interactome studies suggest TTYH3 may interact with numerous proteins including CDKAL1, LMAN2, DHRS9, P2RY12, and others

Gene ontology (GO) analysis revealed that genes involved with binding functions or constituents of vesicles and membranes account for a high proportion of genes related to TTYH3, suggesting its role in membrane transport functions .

How can TTYH3 expression be effectively manipulated in cancer models?

For researchers interested in studying TTYH3 function in cancer, the following approaches have proven effective:

• Lentiviral-mediated shRNA knockdown - Successfully used to reduce TTYH3 expression by approximately 50% at both mRNA and protein levels in bladder cancer cell lines (J82 and T24)

• Validation methods:

  • RT-qPCR for mRNA expression verification

  • Western blotting for protein expression confirmation

  • Functional assays (proliferation, migration, invasion) to confirm phenotypic effects

When designing knockdown experiments, researchers should carefully select target sequences to ensure specificity and efficiency of TTYH3 suppression while minimizing off-target effects.

What are the challenges in developing specific antibodies against TTYH3?

Developing specific antibodies against TTYH3 presents several challenges:

• Membrane protein complexity - As a multi-pass transmembrane protein, TTYH3 has limited exposed epitopes that can be targeted by antibodies

• Isoform variation - With up to four isoforms reported, developing antibodies that either recognize all isoforms or distinguish between specific isoforms requires careful epitope selection

• Cross-reactivity with other Tweety family members - TTYH1, TTYH2, and TTYH3 share sequence homology, potentially leading to cross-reactivity

• Post-translational modifications - N-glycosylation and potentially other modifications may affect epitope recognition

When selecting or developing antibodies, researchers should prioritize validation through multiple techniques including western blotting with positive and negative controls, immunohistochemistry with appropriate tissue samples, and ideally, testing in knockout or knockdown models.

How does TTYH3 relate to other Tweety family members in research applications?

TTYH3 is part of the Tweety protein family that includes TTYH1, TTYH2, and TTYH3. These family members share structural similarities but may have distinct functions and expression patterns:

• TTYH1 has been found to be a favorable prognostic biomarker in ovarian cancer, in contrast to TTYH3 which predicts poor prognosis

• The transmembrane domains of Tweety proteins are relatively conserved among family members, while C-terminal regions show greater variation

• Different Tweety family members appear to interact with distinct sets of proteins, suggesting diverse functional roles

Researchers studying one Tweety family member should consider the potential redundancy or complementary functions of other family members, especially when interpreting knockout or knockdown phenotypes.

How do post-translational modifications affect TTYH3 function?

TTYH3 undergoes N-glycosylation as a key post-translational modification . While the specific impact of N-glycosylation on TTYH3 function has not been fully characterized, this modification typically affects:

• Protein folding and stability

• Membrane trafficking and localization

• Channel function and regulation

• Immune recognition and protein-protein interactions

Researchers investigating TTYH3 function should consider how glycosylation status might affect experimental outcomes, particularly in different cell types or tissue contexts where glycosylation machinery may vary.

How does TTYH3 expression correlate with treatment response in cancer?

Emerging evidence suggests TTYH3 expression may predict treatment responses in cancer patients:

• In ovarian cancer, high expression of TTYH3 predicted unfavorable survival rates in patients treated with platinum, taxol, or platinum + taxol chemotherapy regimens

• In contrast, TTYH1 (another Tweety family member) was associated with better prognosis in ovarian cancer patients receiving these same chemotherapy regimens

This suggests TTYH3 expression analysis might help predict chemotherapy efficacy and inform treatment decisions in certain cancer types.

What is the relationship between TTYH3 and TP53 mutations in cancer?

TTYH3 shows interesting interactions with TP53 mutation status in cancer:

• Enhanced TTYH3 expression was related to worse prognosis specifically in TP53-mutated ovarian cancer patients

• In contrast, up-regulated expression of TTYH1 was associated with favorable progression-free survival in ovarian cancer patients with TP53 mutations

Researchers have speculated that TTYH1 and TTYH3 may regulate TP53-mediated tumorigenesis, potentially through binding to the promoter region of TP53 . This relationship warrants further investigation and may have implications for cancer treatment strategies targeting TP53-mutated tumors.

What are emerging areas of TTYH3 research?

Based on current findings, several promising research directions for TTYH3 include:

• Exploration of TTYH3 as a therapeutic target in cancer, particularly in treatment-resistant tumors

• Investigation of the structural basis for TTYH3 chloride channel function

• Detailed mapping of TTYH3 protein interaction networks in normal and disease states

• Development of isoform-specific reagents and approaches to distinguish the roles of different TTYH3 variants

• Exploration of TTYH3's potential as a biomarker for cancer diagnosis, prognosis, and treatment response prediction

What methodological advances would enhance TTYH3 research?

Future advances that would benefit TTYH3 research include:

• Improved structural characterization techniques to resolve the debate about transmembrane domain number and organization

• Development of highly specific small molecule inhibitors targeting TTYH3

• Creation of conditional knockout mouse models to study tissue-specific TTYH3 functions

• Advanced imaging techniques for visualizing TTYH3 dynamics in living cells

• Single-cell analysis methods to examine TTYH3 expression heterogeneity in complex tissues

Such methodological advances would address current limitations in TTYH3 research and open new avenues for understanding its biological functions and disease relevance.